Single-molecule analysis of DNA interrogation by Cas9 and Cas12a on supercoiled DNA

Cas9 and Cas12a are CRISPR endonucleases that interrogate DNA sequences by forming an R-loop structure containing an RNA:DNA heteroduplex before cleaving a target site that matches a guide RNA. The sequence specificity of R-loop formation and cleavage has enabled wide applications in genetic engineering. R-loop substeps are affected by mismatches with the guide RNA and by target DNA topology, which must be understood to arrive at mechanochemical descriptions of the mechanisms underlying specificity in different CRISPR endonucleases.