Cas9
From Proteopedia
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.
Microbiologist and 2020 Nobel laureate Emanuelle Charpentier, from Max Planck Institute for Infection Biology in Berlin, is one of the co-inventors of the groundbreaking
This video, by Paul Andersen, explains how the CRISPR/Cas immune system
was identified in bacteria and how the CRISPR/Cas9 system was developed to edit genomes.
a powerful new technology with many applications in biomedical research,
including the potential to treat human genetic disease.
Articles in Proteopedia concerning Cas9 include:
3D structures of Cas9
See Endonuclease 3D structures.
STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA
Cas9 OverviewCRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats 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. Cas9 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 Protospacer Adjacent Motif 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 Cas9 are the REC lobe (residues 41–425) and NUC lobe (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 [1]; each of these domains are involved in how Cas9 cuts the target DNA [2],[3],[4]. These lobes are connected by an arginine rich bridge helix (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 heteroduplexThe recognition of the sgRNA-target heteroduplex 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 sgRNA (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 A-form conformation, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the sugar-phosphate backbone (the hydrogen bonds are shown as black dashes). The target DNA binds to the REC lobe and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA[1]. Recognition of the PAM sequenceFor the recognition of the PAM sequence, 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 [1]. Recognition of the sgRNA scaffoldThe SaCas9 recognizes the sgRNA scaffold within the REC lobe and WED domain. 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 [1]. Endonuclease Activity of Cas9Finally, RuvC and HNH are involved in endonuclease activity. Binding to the target DNA causes a conformational change in the H-N-H motif[5], 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 [1]. |
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References
- ↑ 1.0 1.1 1.2 1.3 1.4 Nishimasu H, Cong L, Yan WX, Ran FA, Zetsche B, Li Y, Kurabayashi A, Ishitani R, Zhang F, Nureki O. Crystal Structure of Staphylococcus aureus Cas9. Cell. 2015 Aug 27;162(5):1113-26. doi: 10.1016/j.cell.2015.08.007. PMID:26317473 doi:http://dx.doi.org/10.1016/j.cell.2015.08.007
- ↑ Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. Epub 2004 Dec 13. PMID:15596446 doi:10.1074/jbc.M408446200
- ↑ Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014, Mar 14. PMID:24634220 doi:http://dx.doi.org/10.1074/jbc.M113.539726
- ↑ Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb, 13. PMID:24529477 doi:http://dx.doi.org/10.1016/j.cell.2014.02.001
- ↑ Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID:30555184 doi:http://dx.doi.org/10.1017/S0033583518000070
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