Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a

Abstract

Discovered as an adaptive immune system of prokaryotes, CRISPR–Cas provides many promising applications. DNA-cleaving Cas enzymes like Cas9 and Cas12a, are of great interest for genome editing. The specificity of these DNA nucleases is determined by RNA guides, providing great targeting adaptability. Besides this general method of programmable DNA cleavage, these nucleases have different biochemical characteristics, that can be exploited for different applications. Although Cas nucleases are highly promising, some room for improvement remains. New developments and discoveries like base editing, prime editing, and CRISPR-associated transposons might address some of these challenges.

A gate and clamp regulate sequential DNA strand cleavage by CRISPR-Cas12a

“…From the occupation probabilities, we can also suggest a simple free energy diagram for the different states (Figure 2h). The S1, S2 and S3 states could correspond to the previously identified conformation checkpoints that couple R-loop propagation to nuclease activation [8]. The linker and lid interactions may produce the occupancy of the S1 and S2 states, respectively.…”

“…A key difference between Type II-A and Type V-A CRISPR-Cas effectors is that the former use two separate nuclease domains to cut the NTS and TS while the latter use a single RuvC domain that must transition sequentially between the NTS and TS (8). It has been suggested that R-loop asymmetry is exploited by Cas12a to allow downstream DNA breathing that is a key step in providing the single strand TS that can be delivered to the RuvC nuclease active site (30).…”

“…Additionally, Cas12a has been exploited for nucleic acid recognition diagnostics, including detection of SARS-CoV-2 genomic RNA (7). In both systems, RNA-guided DNA recognition occurs by strand separation of a protospacer target to allow Watson-Crick base pairing between the DNA targeted-strand (TS) and the spacer sequence of a CRISPR RNA (crRNA), and the release of a non-targeted strand (NTS) (8). Type II enzymes such as Cas9 employ two nuclease domains with distinct phylogenies, RuvC and HNH, to cut the NTS and TS, respectively (9,10).…”

“…In contrast, Type V CRISPR effectors have a single RuvC domain which for the prototype Cas12a (formerly known as Cpf1) cuts DNA in an obligatory sequential mechanism; NTS cleavage first followed by ~20fold slower TS cleavage (11)(12)(13)(14)(15)(16). However, it is unclear how the Cas12a RuvC domain transitions between cleaving the two strands (17). A better understanding of this mechanism will aid in the design of Cas12a enzymes with improved properties for gene editing and/or diagnostic applications.…”

“…This gap formation may prevent steric occlusion of the open active site to allow subsequent TS binding and allow binding of non-specific ssDNA in trans ("bystander" cleavage), a key activity for nucleic acid detection diagnostics (31). A geometric limitation for TS cleavage is that the target site is displaced (32), in a region of dsDNA downstream of the R-loop, ~25 A˚ from the RuvC active site and in an incorrect orientation for in-line nucleophilic attack without rotation to match the polarity of the NTS (8,11). A suggested model is that downstream dsDNA unwinds, and the released single stranded TS is bent towards RuvC (Supplementary Figure S1) (13).…”