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CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. 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 bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. 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 REC lobe (residues 41–425) and NUC lobe (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 <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. 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.
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. 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 bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. 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 REC lobe (residues 41–425) and NUC lobe (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 <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. 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 ==
== Recognition of the sgRNA-target heteroduplex ==
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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 a 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/5'>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/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><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 a 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 REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.
== Recognition of the PAM sequence ==
== Recognition of the PAM sequence ==
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.

Revision as of 20:11, 10 October 2022

STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA

PDB ID 5axw

Drag the structure with the mouse to rotate

References

  1. ↑ 1.0 1.1 1.2 1.3 1.4 1.5 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
  2. ↑ 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
  3. ↑ 3.0 3.1 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
  4. ↑ 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
  5. ↑ 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
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