- The Question
- The Answer
- CRISPR Background
- Genome Editing
- Cas9 Mechanism
- Molecular Techniques
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) (Jinek et al. 2014; Nishimasu et al. 2014).
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 (Nishimasu et al. 2014). The PAM-Interacting domain confers PAM specificity and is therefore responsible for initiating binding to target DNA (Anders et al. 2014; Jinek et al. 2014; Nishimasu et al. 2014; Sternberg et al. 2014). 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 (Jinek et al. 2014; Nishimasu et al. 2014).
The Cas9 protein remains inactive in the absence of guide RNA (Jinek et al. 2014). 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) (Jinek et al. 2012; Nishimasu et al. 2014). The guide RNA is engineered to have a 5′ end that is complimentary to the target DNA sequence.
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 (Jinek et al. 2014).
Once the Cas9 protein is activated, it stochastically searches for target DNA by binding with sequences that match its protospacer adjacent motif (PAM) sequence (Sternberg et al. 2014). 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 (Mojica et al. 2009). The PAM in Streptococcus pyogenes is 5′-NGG-3′ (Jinek et al. 2012). When the Cas9 protein finds a potential target sequence with the appropriate PAM, the protein will melt the bases immediately upstream of the PAM and pair them with the complementary region on the guide RNA (Sternberg et al. 2014). 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 (Anders et al. 2014) (Figure 4).