Substrate interrogation of the CRISPR-Cas12a endonuclease reveals an unexpected functional plasticity

Abstract

Substrate interrogation of the CRISPR-Cas12a endonuclease reveals an unexpected functional plasticity Currently, Cas9 and Cas12a (also known as Cpf1) are the sole members of a large family of RNA-guided nucleases that have been widely used in genome manipulation and clinical interventions. Current studies on target discrimination by Cas9 and Cas12a nucleases are limited to single-stranded/double-stranded DNA, negatively supercoiled DNA and substrates with mismatch(es) between the RNA-guide and the DNA target-strand. However, much less is known about the full substrate landscape and biological functions of Cas12a nuclease. Knowledge on the mechanistic aspects of CRISPR-associated Cas nucleases is essential not only for genome engineering but also for reducing the risk off-target effects on the genome. Elucidation of substrate specificity of Cas12a would be important for understanding its role in bacterial immunity against invading bacteriophages and plasmids. In this study, we report the characterization of guide-RNA independent binding and cleavage activity of CRISPR-AsCas12a using a variety of branched DNA structures. Purified AsCas12a was found to possess higher affinity towards various branched DNA substrates, as compared to single- and double strand DNA, without the participation of a divalent cation. Importantly, it showed highest binding affinity towards Holliday junction, among all the branched DNA species. In the presence of Mn2+ ion, Cas12a cleaved a variety of branched DNA substrates, including Holliday junction. Mapping of cleavage sites on a Holliday junction substrate revealed random, non-sequence specific mode of DNA cleavage. A glutaraldehyde crosslinking experiment suggests that Cas12a exists, and likely performs DNA binding and cleavage, as a monomer. A point mutation in its RuvC-like domain abrogated its cleavage function, but not DNA binding activity. We found that AsCas12a binds to the crossover point and to each of the four arms of the HJ. This result is compatible with the findings that AsCas12a unwinds the HJ and non-specifically cleaves various branched DNA species in a RNA independent manner through a combination of endo- and exonuclease activities. In line with this, we observed that AsCas12a has an intrinsic RNA-independent, Mn2+-stimulated exonuclease activity that allows it to resect mononucleotides in the 5'-to- 3' direction. Furthermore, AsCas12a catalyses RNA-independent dsDNA cleavage (but not ssDNA) in the presence of Mn2+. Strikingly, AsCas12a variant harbouring a point mutation (D908A) in the RuvC-I domain and truncation variants lacking the RuvC-III domain or both RuvC-II and RuvC-III domains while retaining nearly the wild-type levels of HJ-binding activity showed reduced (△RuvC-III variant) and abrogation of (RuvC-ID908A and △RuvC-III+RuvC-II variants) DNA cleavage activities, respectively. An anti-CRISPR protein, AcrIIA4, inhibits the DNA cleavage activities of Cas12a on both unbranched and branched DNA substrates, but does not inhibit Cas12a-DNA complex formation. Altogether, these results uncover a broad range of DNA cleavage activities, which has implications in genome editing application and recognition of foreign DNA and cleavage.