Sandbox Reserved 1750: Difference between revisions

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 NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The REC lobe 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 (Scene of 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 (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (Scene of 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 (scene of 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 (scene of domain), 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.