Cas9: Difference between revisions

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 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. <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 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 <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 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.

The 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 <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/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" />.