https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Brett+M.+Thumm 2026-09-24T13:01:48Z User contributions MediaWiki 1.43.8 https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2497013 2015-11-03T01:38:27Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' side='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' &gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The &lt;scene name='71/714945/Rec-2_domain/1'&gt;REC-2&lt;/scene&gt; domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The &lt;scene name='71/714945/Ruvc_domain/1'&gt;RuvC nuclease&lt;/scene&gt; cleaves the non-complementary, single stranded DNA. This nuclease is active through the use of four catalytic residues shown in the previous scene. The &lt;scene name='71/714945/Hnh_domain/3'&gt;HNH domain&lt;/scene&gt; is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The four catalytic residues of this nuclease domain are highlighted in red. The &lt;scene name='71/714945/Pi/1'&gt;PI&lt;/scene&gt; is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496821 2015-10-28T02:07:09Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' side='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' &gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The &lt;scene name='71/714945/Rec-2_domain/1'&gt;REC-2&lt;/scene&gt; domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The &lt;scene name='71/714945/Ruvc_domain/1'&gt;RuvC nuclease&lt;/scene&gt; cleaves the non-complementary, single stranded DNA. This nuclease is active through the use of four catalytic residues shown in the previous scene. The &lt;scene name='71/714945/Hnh_domain/3'&gt;HNH domain&lt;/scene&gt; is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The four catalytic residues of this nuclease domain are highlighted in red. The &lt;scene name='71/714945/Pi/1'&gt;PI&lt;/scene&gt; is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496820 2015-10-28T02:04:42Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' side='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' &gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The &lt;scene name='71/714945/Rec-2_domain/1'&gt;REC-2&lt;/scene&gt; domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The &lt;scene name='71/714945/Ruvc_domain/1'&gt;RuvC nuclease&lt;/scene&gt; cleaves the non-complementary, single stranded DNA. This nuclease is active through the use of four catalytic residues shown in the previous scene. The &lt;scene name='71/714945/Hnh_domain/3'&gt;HNH domain&lt;/scene&gt; is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The &lt;scene name='71/714945/Pi/1'&gt;PI&lt;/scene&gt; is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496776 2015-10-27T14:20:30Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' side='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' &gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The &lt;scene name='71/714945/Rec-2_domain/1'&gt;REC-2&lt;/scene&gt; domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The &lt;scene name='71/714945/Ruvc_domain/1'&gt;RuvC nuclease&lt;/scene&gt; cleaves the non-complementary, single stranded DNA. This nuclease is active through the use of four catalytic residues shown in the previous scene. The &lt;scene name='71/714945/Hnh_domain/2'&gt;HNH domain&lt;/scene&gt; is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The &lt;scene name='71/714945/Pi/1'&gt;PI&lt;/scene&gt; is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496646 2015-10-22T17:43:42Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The &lt;scene name='71/714945/Rec-2_domain/1'&gt;REC-2&lt;/scene&gt; domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The &lt;scene name='71/714945/Ruvc_domain/1'&gt;RuvC nuclease&lt;/scene&gt; cleaves the non-complementary, single stranded DNA. This nuclease is active through the use of four catalytic residues shown in the previous scene. The &lt;scene name='71/714945/Hnh_domain/1'&gt;HNH domain&lt;/scene&gt; is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The &lt;scene name='71/714945/Pi/1'&gt;PI&lt;/scene&gt; is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496645 2015-10-22T17:42:04Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The &lt;scene name='71/714945/Rec-2_domain/1'&gt;REC-2&lt;/scene&gt; domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The &lt;scene name='71/714945/Ruvc_domain/1'&gt;RuvC nuclease&lt;/scene&gt; cleaves the non-complementary, single stranded DNA. This nuclease is active through the use of four catalytic residues shown in the previous scene. The &lt;scene name='71/714945/Hnh_domain/1'&gt;HNH domain&lt;/scene&gt; is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The &lt;scene name='71/714945/Pi/1'&gt;PI&lt;/scene&gt; is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> &lt;/StructureSection&gt;<br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496644 2015-10-22T17:33:21Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The &lt;scene name='71/714945/Rec-2_domain/1'&gt;REC-2&lt;/scene&gt; domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The &lt;scene name='71/714945/Ruvc_domain/1'&gt;RuvC nuclease&lt;/scene&gt; cleaves the non-complementary, single stranded DNA. This nuclease is active through the use of four catalytic residues shown in the previous scene. The &lt;scene name='71/714945/Hnh_domain/1'&gt;HNH domain&lt;/scene&gt; is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> &lt;/StructureSection&gt;<br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496643 2015-10-22T17:19:54Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The &lt;scene name='71/714945/Rec-2_domain/1'&gt;REC-2&lt;/scene&gt; domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The &lt;scene name='71/714945/Ruvc_domain/1'&gt;RuvC nuclease&lt;/scene&gt; cleaves the non-complementary, single stranded DNA. This nuclease is active through the use of four catalytic residues shown in the previous scene. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> &lt;/StructureSection&gt;<br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496642 2015-10-22T17:04:57Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The &lt;scene name='71/714945/Rec-2_domain/1'&gt;REC-2&lt;/scene&gt; domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> &lt;/StructureSection&gt;<br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496641 2015-10-22T16:59:16Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/2'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> &lt;/StructureSection&gt;<br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496640 2015-10-22T16:47:37Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The &lt;scene name='71/714945/Rec-1_domain/1'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> &lt;/StructureSection&gt;<br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496639 2015-10-22T16:46:56Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The scene name='71/714945/Rec-1_domain/1'&gt;REC-1&lt;/scene&gt; domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> &lt;/StructureSection&gt;<br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2496638 2015-10-22T16:32:00Z

<p>Brett M. Thumm: </p> <hr /> <div>== Function ==<br /> &lt;StructureSection load='4un3' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long &lt;scene name='71/714945/Alpha_helix_bridge/1'&gt;α-helix bridge&lt;/scene&gt;, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> &lt;/StructureSection&gt;<br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2493385 2015-10-13T17:56:24Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/12'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2493384 2015-10-13T17:48:23Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/5'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2493383 2015-10-13T17:46:12Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/3'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2493382 2015-10-13T17:42:08Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/4'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2493287 2015-10-13T13:16:12Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/11'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/3'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491563 2015-10-06T16:12:00Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/10'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/3'&gt;domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491562 2015-10-06T16:10:02Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/10'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease, these are the essential &lt;scene name='71/714945/Lobes_domain_of_cas9/1'&gt;Domains&lt;/scene&gt;. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491561 2015-10-06T15:25:53Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/10'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491560 2015-10-06T15:18:58Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease &lt;scene name='71/714945/Pam_interaction/9'&gt;Cas9&lt;/scene&gt; has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491559 2015-10-06T14:59:59Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='4un3 of Streptococcus pyogenes' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease Cas9 has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491558 2015-10-06T14:54:05Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> [[Image:4un3 labeled.png|right|390px]]<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease Cas9 has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491557 2015-10-06T14:52:40Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease Cas9 has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] [[Image:4un3 labeled.png|right|390px]]<br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491556 2015-10-06T14:51:52Z

<p>Brett M. Thumm: </p> <hr /> <div>&lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease Cas9 has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. &lt;ref&gt;PMID:24906146&lt;/ref&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA. This structure has been shown to be conserved through Cas9 proteins. The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices. The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.&lt;ref&gt;PMID:24529477&lt;/ref&gt;<br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9 &lt;ref&gt;PMID:25079318&lt;/ref&gt;. The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]] [[Image:4un3 labeled.png|right|390px]]<br /> <br /> <br /> <br /> <br /> <br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491555 2015-10-06T14:37:09Z

<p>Brett M. Thumm: </p> <hr /> <div><br /> &lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA (CITE 2). This structure has been shown to be conserved through Cas9 proteins (http://www.cell.com/abstract/S0092-8674(14)00156-1 ). The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices (CITE CELL). The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.<br /> <br /> <br /> <br /> == Function ==<br /> [[Cas9]] is the RNA-guided [[DNA endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, [[Cas9]] requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease Cas9 has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences.<br /> <br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9(CITE #1). The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491554 2015-10-06T14:34:09Z

<p>Brett M. Thumm: </p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> &lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> <br /> <br /> == Structural highlights ==<br /> <br /> Cas9 is a structurally bilobed, containing specific domains for the recognition of target DNA and nucleases to cleave DNA strands. Each of these lobes contain three major units essential for a functional endonuclease. <br /> <br /> The recognition lobe (REC) contains a long bridge helix, the REC-1 domain and the REC-2 domain. It is also the least conserved lobe through the types of Cas9. The long α-helix bridge, which is arginine-rich, is essential for recognizing single guide RNA-DNA complexes on target DNA (CITE 2). This structure has been shown to be conserved through Cas9 proteins (http://www.cell.com/abstract/S0092-8674(14)00156-1 ). The REC-1 domain contains 25 α-helixes and two β-sheets, and is crucial to the function of Cas9 by recognizing a specific motif termed the repeat:anti-repeat region of the single guide RNA and DNA complex. The REC-2 domain contains six α-helix’s in a bundle but there is no current understanding of its function.<br /> <br /> The nuclease lobe (NUC) contains a RuvC domain, HNH domain and the PAM-interacting domain (PI). The RuvC domain is comprised of three RuvC motifs that are made up of two-stranded antiparallel β-sheets, and six-stranded β-sheets, which are flanked by nine α-helices (CITE CELL). The RuvC nuclease cleaves the non-complementary, single stranded DNA. The HNH domain is composed of a two-stranded antiparallel β-sheet which is flanked by four α-helixes and cleaves the complementary strand of target DNA. The PI is made up of seven α-helixes and numerous strand-varying antiparallel β-sheets which recognize the PAM sequence on the non-complementary target DNA strand.<br /> <br /> <br /> <br /> == Function ==<br /> Cas9 is the RNA-guided DNA endonuclease used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, Cas9 requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication. The CRISPR-associated endonuclease Cas9 has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences.<br /> <br /> <br /> <br /> == DNA Interaction ==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand, which constitues the &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction at the PAM sequence&lt;/scene&gt;. This canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, therefore, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9(CITE #1). The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding atoms, some of which are accomplished through water molecules, and ionic interactions. A phosphate lock loop provides local strand separation upstream of the PAM sequence when a Lysine and Serine residue stabilize target DNA. The PAM sequence recognition of Cas9 is an integral function of its specific binding, subsequent base pair melting and cleavage of target DNA<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> <br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491553 2015-10-06T14:26:04Z

<p>Brett M. Thumm: </p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> &lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> <br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction with PAM sequence&lt;/scene&gt;<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491552 2015-10-06T14:22:49Z

<p>Brett M. Thumm: </p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> &lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> &lt;scene name='71/714945/Pam_interaction/8'&gt;Cas9 interaction with PAM &gt;<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491551 2015-10-06T14:19:00Z

<p>Brett M. Thumm: </p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> &lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> &lt;scene name='71/714945/Pam_interaction/8'&gt;TextToBeDisplayed&lt;/scene&gt;<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]<br /> <br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491550 2015-10-06T14:00:54Z

<p>Brett M. Thumm: </p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> &lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> &lt;scene name='71/714945/Pam_interaction/5'&gt;TextToBeDisplayed&lt;/scene&gt;<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]<br /> <br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491530 2015-10-06T13:06:40Z

<p>Brett M. Thumm: </p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> &lt;Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> &lt;scene name='71/714945/Pam_interaction/1'&gt;CAS9&lt;/scene&gt;<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]<br /> <br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491529 2015-10-06T13:05:54Z

<p>Brett M. Thumm: </p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> &lt;Structure load='&lt;scene name='71/714945/Pam_interaction/1'&gt;CAS9&lt;/scene&gt;' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]<br /> <br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491521 2015-10-06T12:42:03Z

<p>Brett M. Thumm: </p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> &lt;Structure load='Insert PDB code or filename here' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]<br /> <br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Cas9_Sandbox&diff=2491506 2015-10-06T12:18:21Z

<p>Brett M. Thumm: New page: ==Your Heading Here (maybe something like &#039;Structure&#039;)== &lt;StructureSection load=&#039;1stp&#039; size=&#039;340&#039; side=&#039;right&#039; caption=&#039;Caption for this structure&#039; scene=&#039;&#039;&gt; This is a default text for you...</p> <hr /> <div>==Your Heading Here (maybe something like 'Structure')==<br /> &lt;StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> This is a default text for your page '''Cas9 Sandbox'''. Click above on '''edit this page''' to modify. Be careful with the &amp;lt; and &amp;gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia &lt;ref&gt;DOI 10.1002/ijch.201300024&lt;/ref&gt; or to the article describing Jmol &lt;ref&gt;PMID:21638687&lt;/ref&gt; to the rescue.<br /> <br /> == Function ==<br /> <br /> &lt;Structure load='Insert PDB code or filename here' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to &lt;scene name=&quot;/12/3456/Sample/1&quot;&gt;color&lt;/scene&gt; by Group, and another to make &lt;scene name=&quot;/12/3456/Sample/2&quot;&gt;a transparent representation&lt;/scene&gt; of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Thumm_prac_sandbox&diff=2491488 2015-10-05T03:41:18Z

<p>Brett M. Thumm: </p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> &lt;Structure load='Insert PDB code or filename here' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /&gt;<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Thumm_prac_sandbox&diff=2491487 2015-10-05T03:39:20Z

<p>Brett M. Thumm: New page: Cas9 is the RNA-guided DNA endonuclease used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks ...</p> <hr /> <div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br /> <br /> The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm https://proteopedia.org/index.php?title=Elizeu/sandbox/citocromo_c&diff=2491475 2015-10-01T20:03:37Z

<p>Brett M. Thumm: </p> <hr /> <div>==Function==<br /> [[Cas9]] is the RNA-guided DNA [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response. Three different types of CRISPR mechanisms have been discovered, however, only type II CRISPR systems are heavily researched. In vivo, Cas9 requires CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA)to guide the endonuclease toward invading DNA based on the complementary sequence recognition of these RNAs. Cas9 then targets and cleaves foreign DNA to interfere with viral replication.<br /> <br /> The CRISPR-associated endonuclease Cas9 has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to perform the function of crRNA-tracRNA complex to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences.<br /> <br /> [[Image:Layout for schematic and structure with structure.png|660px]]<br /> <br /> ==Interaction with DNA==<br /> Target DNA contains a protospacer adjacent motif on the non-complementary strand. This is a canonical sequence of 5’-NGG-3’ is recognized by Cas9 and is essential for genetic interference and editing, it will not cleave the target sequence if the PAM sequence is absent. The double Guanine of the non-complementary sequence strand interacts via hydrogen bonding from the major groove through two conserved arginine residues on the carboxy-terminus of Cas9(CITE #1). The minor groove of the PAM sequence interacts with a serine, through a hydrogen bridge to the last guanine, and lysine residue on the complementary target strand of the middle guanine. <br /> <br /> The deoxyribose-phosphate backbone of the non-complementary strand is arranged in close proximity to various hydrogen bonding, some of which are accomplished through water molecules, and ionic interactions. (MMMOOORREE)<br /> <br /> Articles in Proteopedia concerning Cas9 include:<br /> * any?<br /> <br /> ==3D structures of Cas9==<br /> <br /> ===''Streptococcus pyogenes'' Cas9===<br /> <br /> * [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> [[Image:4un3 labeled.png|right|390px]]<br /> * [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br /> * [[4cmp]]<br /> * [[4cmq]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''S. pyogenes'' Cas9<br /> <br /> === ''Actinomyces naeslundii'' Cas9=== <br /> *[[4oge]]<br /> *[[4ogc]] - Mn&lt;sup&gt;2+&lt;/sup&gt;-bound ''A.s naeslundii'' Cas9<br /> <br /> ==See Also==<br /> [[HNH endonuclease]] &lt;br/&gt;<br /> <br /> <br /> <br /> == References ==<br /> &lt;references/&gt;<br /> <br /> [[Category: Crispr]]<br /> [[Category: Crispr-associated]]<br /> [[Category: endonuclease]]</div>

Brett M. Thumm