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. 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 nucleic acids present causing a conformational change in the HNH locking the HNH 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 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 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 in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA [1].
