Spacers are used to target foreign nucleic acids containing sequences complementary to the spacer, termed protospacers, for degradation in a sequence-dependent manner, the process is known as interference which is the last stage of CRISPR immunity following by spacer acquisition and crRNA processing. During interference, invading nucleic acids detected by base pairing with crRNA (CRISPR RNA) are targeted for degradation by an interference nuclease. In type I CRISPR-Cas systems this is the HD metal-dependent nuclease domain of Cas3, which is recruited to Cascade rather than being an integral component. Type II systems use Cas9 as the sole interference protein with the HNH and RuvC nuclease domains cleaving the complementary and non-complementary strands of the R-loop respectively.
The principle of target interference by CRISPR-Cas systems is that crRNA bound to Cas protein(s) locate the corresponding protospacer to trigger degradation of the target nucleic acids. Interference in type I and III systens is facilitated by a large, multi-subunit ribonucleoprotein complex. The targeting complex in type I CRISPR-Cas systems is called Cascade (CRISPR-associated complex for antiviral defense), which is formed by Cse1, Cse2, Cas7, Cas5, Cas6e subunits in E. coli (Type I-E) and Csy1, Csy2, Csy3 and Cas6f in Pseudomonas aeruginosa (Type I-F). Cascade locates the target DNA but the nucleolytic cleavage of the target DNA is carried out by a Cas3 family exonuclease. Cas3 can be recruited by Cascade upon target binding or, in the case of Type I-A, be a permanent part of Cascade. In the Type I-A system the Cas3 nuclease and helicase domains are encoded as separate genes. In all Type I systems the two domains act together to processively degrade the double stranded DNA target.
Fig 1. Mechanism of crRNA processing and DNA interference in the three types of CRISPR-Cas systems.
In Type I and II systems, interference requires the presence of a PAM sequence and perfect protospacer-crRNA complementarity in the so-called "seed region", located adjacent to the PAM. The presence of a PAM triggers "non-self activation", which prevents the systems from attacking its own CRISPR locus. The "seed" sequence is a 6- to 8-nucleotide sequence within the guide crRNA which is strictly complementary to the target sequence, and enthalpically drives complementary base-pairing between the crRNA and the target, and likely R-loop formation. The binding of the crRNA to the target nucleic acids causes conformational changes in Cascade and the target DNA that could be the trigger for Cas3 recruitment. Cas3 then nicks the target DNA and proceeds with progressive degradation of the target while Cascade presumably dissociates and is ready for action again.
Type II systems require only the Cas9 protein for interference but unlike Type I and III systems it needs not just crRNA, but also tracrRNA, a small RNA that shares partial complementarity with CRISPR repeats, bound to Cas9 to perform target recognition and degradation. The structures of Cas9, a single large multidomain protein, from S. pyogenes and Actinomyces naeslundii reveal separate lobes for target recognition and nuclease activity, accommodating the crRNA-DNA heteroduplex in a positively charged groove at their interface. The recognition lobe is important for binding crRNA and target DNA, and the nuclease lobe contains two independent domains, HNH domain and RuvC domain, which cleaves the complementary and non-complementary strands of the target, respectively. Recent studies have established that formation of a complex with crRNA:tracrRNA drives Cas9 conformation changes that direct target DNA binding and cleavage, in a PAM-dependent manner.
Once Cas9 binds its guide RNA, the complex is ready to search for complementary target DNA sites. Target search and recognition require both complementary base pairing between the 20-nt spacer sequence and a protospacer in the target DNA, as well as the presence of conserved PAM sequence. Once Cas9 has found a target site with the appropriate PAM, it triggers local DNA melting at the PAM-adjacent nucleation site, followed by RNA strand invasion to form an RNA-DNA hybrid and a displaced DNA strand (termed R-loop) from PAM-proximal to PAM-distal ends. Upon PAM recognition and subsequent RNA-DNA duplex formation, Cas9 is activated for DNA cleavage. Each of the Cas9 nuclease domain cleaves one strand of the target dsDNA at a specific site 3bp from the PAM sequence to produce a predominantly blunt-ended DSB.
And then the DSB is repaired by host-mediated DNA repair mechanisms subsequently, such as NHEJ and HDR. HDR allows an exogenous "donor" sequence to be provided to the cell and swapped into the genome, causing specific changes in the sequence. NHEJ is a repair mechanism that is useful when disruption of any sort at the DSB site will give the desired effect. Among the repair pathways for the DSBs generated by CRISPR–Cas9, HDR seems to be a precise and high-fidelity mechanism for error-fixing, but NHEJ could result in unwanted insertions or deletions. One of the major challenges of gene editing is to induce HDR mechanisms and reduce the degree of NHEJ.
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