Kleinstiver, B.P. et al. Nat. Biotechnol. 34, 869–874 (2016).

Most genome editing with the CRISPR system to date has been carried out with the type II nuclease Cas9 from Streptococcus pyogenes. Recently, another type II nuclease, Cpf1, was introduced with features that SpCas9 is lacking. Cpf1 requires only a 42-nt short CRISPR RNA (crRNA) to find its target, instead of the ∼100-nt guide RNA for SpCas9, and it recognizes a protospacer-adjacent motif (PAM) that is 5′ instead of 3′ of the target site. Kleinstiver et al. have now compared the two nucleases and found Cpf1 to be efficient and highly specific in human cells, with rare off-target cleavage. Systematically introducing mutations into the crRNA showed that mismatches at the 5′ end are not tolerated, whereas mismatches at the very 3′ end of the crRNA have less of an effect on cleavage. This high specificity makes Cpf1 a candidate for therapeutic applications down the line.