Investigating the Mechanism of DNA Recognition by a CRISPR-Cas12a Nuclease

Abstract

Clustered-Regularly-Interspersed-Short-Palindromic-Repeats (CRISPR) and CRISPR-associated (Cas) proteins provide an efficient means to target genomic DNA sequences in a large variety of organisms, leading to a revolution in genome engineering. Both the class 2 type II Cas9, which has been widely used in genome editing, and the recently discovered class 2 type V Cas12a (Cpf1), use an effector complex comprising of a single protein activated by CRISPR-encoded small RNA(s) (crRNA) to cleave double-stranded DNA at specific sites. However, Cas12a differs from Cas9 in key features of effector protein, crRNA, and elements of recognition in target DNA, and has been reported to show a higher degree of discrimination against mis-matches between the guide RNA and the DNA. To elucidate the mechanism of DNA recognition by Cas12a, which may underlie its enhanced substrate specificity, we are using a combination of cleavage, binding and competition assays to examine Cas12a recognition a variety of DNA constructs. The data showed for topologically flexible DNA substrates, including those containing unpaired protospacer segments, were bound at the cognate Cas12a active site even in the absence of pairing between the RNA guide and the DNA target strand. However, these substrates were not cleaved by the enzyme, suggesting that Cas12a employs proof-reading mechanisms subsequent to initial DNA binding to ensure proper cleavage. The results have implications on the mechanisms of Cas12a DNA recognition, as well as in applications that rely on CRISPR binding to DNA, such as transcription regulation and base editing.