CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA

The CRISPR-associated protein Cas12a (previously known as Cpf1), which is an endonuclease from the type V-A CRISPR system, has been applied in both in vivo genome editing and in vitro DNA assembly. 1 , 2 , 3 Cas12a is guided by a single CRISPR RNA (crRNA) with a T-rich protospacer adjacent motif (PAM) sequence to cleave double-stranded DNA (dsDNA) targets, generating sticky ends. Different from Cas9, Cas12a cleaves both the target and non-target strands of a targeted dsDNA by a single active site in the RuvC catalytic pocket 4 , 5 , 6 (Supplementary information, Figure S12a ). Besides, Cas12a also processes precursor crRNAs to generate mature crRNAs. 7 However, the cleavage activity of Cas12a on single-stranded DNA (ssDNA) targets is less understood.