Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

Abstract

ABSTRACT CRISPR-Cas12a (Cpf1) are RNA-guided nuclease effectors of acquired immune response that act in their native organisms by cleaving targeted DNA sequences. Like CRISPR-Cas9 RNA-guided DNA targeting enzymes, Cas12a orthologs have been repurposed for genome editing in non-native organisms and for DNA manipulation in vitro . Recent studies have shown that activation of Cas12a via guide RNA-target DNA pairing causes multiple turnover, non-specific ssDNA degradation in trans , after single turnover on-target cleavage in cis . We find that the non-specific trans nuclease activity affects RNA and dsDNA in addition to ssDNA, an activity made more evident by adjustment of reaction buffer composition. The magnitude of the trans nuclease activity varies depending on features of the guide RNA being used, specifically target sequence composition and length. We also find that the magnitude of trans nuclease activity varies between the three most well-studied Cas12a orthologs and that the Cas12a from Lachnospiraceae bacterium ND2006 appears to be the most active.