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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 by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. 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 are the (residues 41–425) and (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 ^[1]. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA ^[2][3][4]. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site ^[5]. 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

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 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 (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 , so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the (the hydrogen bonds are shown as black dashes). The target DNA binds to the for the proper conformation for base paring between the target DNA and sgRNA^[1].

Recognition of the PAM sequence

For the recognition of the , 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 scaffold

The SaCas9 recognizes the sgRNA scaffold within the . 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 Cas9

Finally, are involved in endonuclease activity. 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 confromational change is not present in the crystal structure, therefore the HNH appears to be far from the target DNA. 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].