Sandbox Reserved 1750: Difference between revisions

== 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 clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as 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). 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 NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition 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>. 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 ==