Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

Cas12a (formerly Cpf1) is an RNA-guided endonuclease in the CRISPR-Cas immune system that can be easily programmed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity , making it an ideal candidate for precise genome editing applications. This specificity is contradictory to the natural role of Cas12a as an immune effector against rapidly evolving phages. However, the native cleavage specificity and activity remains to be fully understood. We employed high-throughput cleavage assays to determine and compare the native specificities of three Cas12a orthologs. Surprisingly, we observed pervasive nicking of randomized target libraries, with strong nicking activity observed against targets with up to four mismatches. Nicking and cleavage activities are dependent on mismatch type and position, and vary depending on the Cas12a ortholog and crRNA sequence. Our high-throughput and biochemical analysis further reveal that Cas12a has robust activated non-specific nicking and weak non-specific dsDNA degradation activity in trans. Together, our findings reveal Cas12a cleavage activities that could be beneficial in the context of bacterial CRISPR-Cas immunity but may be detrimental for genome editing technology.