Sandbox Reserved 1750: Difference between revisions

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The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.

== Recognition of the sgRNA-target heteroduplex ==

The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.

The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.

== Recognition of the PAM sequence ==

For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.

Sandbox Reserved 1750: Difference between revisions

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 The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.
 == Recognition of the sgRNA-target heteroduplex ==
-The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.
+The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.
 == Recognition of the PAM sequence ==
 For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.