Understanding the mechanism of CRISPR is crucial for researchers. The key step in editing an organism's genome is selective targeting of a specific sequence of DNA. Two biological macromolecules, the Cas9 protein and guide RNA, interact to form a complex that can identify target sequences with high selectivity. The Cas9 protein is responsible for locating and cleaving target DNA, both in natural and in artificial CRISPR-Cas systems. The Cas9 protein has six domains, REC I, REC II, Bridge Helix, PAM Interacting, HNH and RuvC (Figure 1) [1,2].
The Rec I domain is the largest and is responsible for binding guide RNA. The role of the REC II domain is not yet well understood. The arginine-rich bridge helix is crucial for initiating cleavage activity upon binding of target DNA . The PAM-Interacting domain confers PAM specificity and is therefore responsible for initiating binding to target DNA [1,2,3,4]. The HNH and RuvC domains are nuclease domains that cut single-stranded DNA. They are highly homologous to HNH and RuvC domains found in other proteins [1,2].
The Cas9 protein remains inactive in the absence of guide RNA . In engineered CRISPR systems, guide RNA is comprised of a single strand of RNA that forms a T-shape comprised of one tetraloop and two or three stem loops (Figure 2) [5,2]. The guide RNA is engineered to have a 5′ end that is complimentary to the target DNA sequence.
Fig 1. Cas9 Protein. The Cas9 protein is comprised of six domains: Rec I, Rec II, Bridge Helix, RuvC, HNH, and PAM Interacting. Domains are shown in schematic, crystal, and map form. (original figure) (crystal image rendered from PDB: 4CMP .)
Fig 2. Engineered Guide RNA. Target complimentary region is shown in red. (crystal image rendered from PDB: 4UN3 .)
Engineered guide RNA is a single strand of RNA. It forms one tetraloop and two or three stem loops (three shown). This artificial guide RNA binds to the Cas9 protein and, upon binding, induces a conformational change in the protein (Figure 3). The conformational change converts the inactive protein into its active form. The mechanism of the conformational change is not completely understood, but Jinek and colleagues hypothesize that steric interactions or weak binding between protein side chains and RNA bases may induce the change .
Once the Cas9 protein is activated, it stochastically searches for target DNA by binding with sequences that match its protospacer adjacent motif (PAM, yellow stars) sequence . A PAM is a two- or three-base sequence located within one nucleotide downstream of the region complementary to the guide RNA. PAMs have been identified in all CRISPR systems, and the specific nucleotides that define PAMs are specific to the particular category of CRISPR system . The PAM in Streptococcus pyogenes is 5′-NGG-3′ .
When the Cas9 protein finds a potential target sequence with the appropriate PAM, the protein:guide RNA complex will melt the bases immediately upstream of the PAM and pair them with the target complementary region on the guide RNA . If the complementary region and the target region pair properly, the RuvC and HNH nuclease domains will cut the target DNA after the third nucleotide base upstream of the PAM  (Figure 4).
Fig 3. Activation of Cas9 protein by guide RNA binding. Binding of the guide RNA induces a conformational change in the Cas9 protein. (original figure) (crystal image rendered from PDB: 4UN3 .)
Fig 4. Target DNA binding and cleavage by Cas9. (original figure) (crystal images: Lower left and right rendered from PDB: 4UN3; Upper left rendered from PDB: 4CMP .)
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4. Sternberg S.H et al. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature. 507(7490): 62–67.
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