The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multiprotein 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 Acidominococcus 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. ; United States. Dept. of Energy (Computational Science Graduate Fellowship) ; National Institute of Mental Health (U.S.) (Grant 1DP1-MH100706) ; Poitras Foundation ; Vallee Foundation ; Simons Foundation ; Paul G. Allen Family Foundation ; New York Stem Cell Foundation ; Robert Metcalfe ; David R. Cheng