View source for Cas9

[[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.

See also [[Cas9 (hebrew)]].

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. 

[[Image:Layout for schematic and structure with structure.png|660px]]



<center><html5media height=“315” width=“560” frameborder="0" allowfullscreen>https://www.youtube.com/embed/TdBAHexVYzc</html5media></center>
<center>Geneticist and 2020 Nobel laureate [http://rna.berkeley.edu/ Jennifer Doudna], from UC Berkeley, is one of the co-inventors of the groundbreaking </center>
<center>new technology for editing genes, called CRISPR-Cas9. The tool allows scientists</center>
<center>to make precise edits to DNA strands, which could lead to treatments for genetic diseases … </center>
<center>but could also be used to create so-called "designer babies."  </center>
<center>Doudna reviews how CRISPR-Cas9 works — and asks the scientific community </center>
<center>to pause and discuss the ethics of this new tool.</center>

<center><br>Microbiologist and 2020 Nobel laureate [https://www.emmanuelle-charpentier-lab.org/ Emanuelle Charpentier], from Max Planck Institute for Infection Biology in Berlin, is one of the co-inventors of the groundbreaking </center>
<center>new technology for editing genes, called CRISPR-Cas9. The tool allows scientists</center>
<center>to make precise edits to DNA strands, which could lead to treatments for genetic diseases … </center>
<center>but could also be used to create so-called "designer babies."  </center>
<center>Charpentier reviews how CRISPR-Cas9 works in [https://www.cnn.com/videos/tech/2016/04/27/crispr-cas9-explainer-natpkg.cnn/video/playlists/fertility-health/ this 2016 talk].<br></center>


<center><html5media height=“315” width=“560” frameborder="0"  allowfullscreen>https://www.youtube.com/embed/MnYppmstxIs</html5media>
<br>This video, by Paul Andersen, explains how the CRISPR/Cas immune system <br>was identified in bacteria and how the CRISPR/Cas9 system was developed to edit genomes.</center>
<br>

<center><html5media height=“315” width=“560” frameborder="0" allowfullscreen>https://www.youtube.com/embed/2pp17E4E-O8</html5media></center>
<center>Movie from the [http://mcgovern.mit.edu/ McGovern Institute for Brain Research at MIT]</center>
<center>This animation depicts the CRISPR-Cas9 method for genome editing: <br>a powerful new technology with many applications in biomedical research, <br>including the potential to treat human genetic disease.</center>

Articles in Proteopedia concerning Cas9 include:
* [[Cas9 Sandbox|Brett  Thumm's Student Project page on Cas9]]<br />
*[[Cas9 (hebrew)]].

==3D structures of Cas9==

See [[Endonuclease 3D structures]].

[[Image:4un3 labeled.png|right|390px]]

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[[Category: Crispr]]
[[Category: Crispr-associated]]
[[Category: endonuclease]]

==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''>
== Cas9 Overview ==
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for '''C'''lustered '''R'''egularly '''I'''nterspaced '''S'''hort '''P'''alindromic '''R'''epeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for '''P'''rotospacer '''A'''djacent '''M'''otif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. 

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). The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA. The NUC (nuclease) lobe contains RuvC, HNH, WED, and PI domains <ref name="Cas9">PMID:26317473</ref>; each of these domains are involved in how Cas9 cuts the target DNA <ref>PMID:15596446</ref>,<ref>PMID:24634220</ref>,<ref>PMID:24529477</ref>.  These lobes are connected by an arginine rich <scene name='58/581940/Linker_helix_nucleotides/1'>bridge helix</scene> (residues 41–73) and a linker loop (residues 426–434).  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 <scene name='58/581940/Heteroduplex/1'>heteroduplex</scene> in Cas9 begins by inserting the heteroduplex 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 <scene name='58/581940/Linker_helix_nucleotides/1'>bridge helix</scene> (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/21'>sugar-phosphate backbone</scene> (the hydrogen bonds are shown as black dashes). 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/22'>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" />.

== Recognition of the sgRNA scaffold ==
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.

== Endonuclease Activity of Cas9 ==
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. Binding to the target DNA causes a conformational change in the [[H-N-H motif]]<ref>PMID:30555184</ref>, a conserved endonuclease structure, named for its characteristic histidine-asparagine-histidine conserved residues.   RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack. A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. This linker conformational change is not present in the crystal structure, therefore the HNH appears to be far from the target DNA and in an inactive state. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.
</structuresection>
==References==


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