Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System

Highlights•CRISPR-Cpf1 is a class 2 CRISPR system•Cpf1 is a CRISPR-associated two-component RNA-programmable DNA nuclease•Targeted DNA is cleaved as a 5-nt staggered cut distal to a 5′ T-rich PAM•Two Cpf1 orthologs exhibit robust nuclease activity in human cellsSummaryThe microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.Graphical abstract