Characterized Cas12a (Cpf1) as alternative to Cas9. Demonstrated staggered overhang cleavage, single-guide architecture, and ability to process its own crRNA array for multiplex editing.
Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system
Cas9 was the only characterized class 2 CRISPR effector, and candidate nuclease Cpf1 had no shown activity. Cpf1 is a single RNA-guided nuclease needing no tracrRNA, cutting DNA via a staggered cut near a T-rich PAM. Two orthologs edited human genomes near Cas9 levels.
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Key findings
Abstract
Highlights d CRISPR-Cpf1 is a class 2 CRISPR system d Cpf1 is a CRISPR-associated two-component RNA- programmable DNA nuclease d Targeted DNA is cleaved as a 5-nt staggered cut distal to a 5 0 T-rich PAM d Two Cpf1 orthologs exhibit robust nuclease activity in human cells Authors Bernd Zetsche, Jonathan S. Gootenberg, Omar O. Abudayyeh, ..., Aviv Regev, Eugene V. Koonin, Feng Zhang Correspondence zhang@broadinstitute.org In Brief Cpf1 is a RNA-guided DNA nuclease that provides immunity in bacteria and can be adapted for genome editing in mammalian cells. Zetsche et al., 2015, Cell 163 , 759–771 October 22, 2015 ª 2015 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2015.09.038 Article Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System Bernd Zetsche, 1,2,3,4,5,10 Jonathan S. Gootenberg, 1,2,3,4,6,10 Omar O. Abudayyeh, 1,2,3,4 Ian M. Slaymaker, 1,2,3,4 Kira S. Makarova, 7 Patrick Essletzbichler, 1,2,3,4 Sara E. Volz, 1,2,3,4 Julia Joung, 1,2,3,4 John van der Oost, 8 Aviv Regev, 1,9 Eugene V. Koonin, 7 and Feng Zhang 1,2,3,4, * 1 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA 2 McGovern Institute for Brain Research 3 Department of Brain and Cognitive Sciences 4 Department of Biological Engineering Massachusetts Institute of Technology, Cambridge, MA 02139, USA 5 Department of Developmental Pathology, Institute of Pathology, Bonn Medical School, Sigmund Freud Street 25, 53127 Bonn, Germany 6 Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA 7 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA 8 Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, Netherlands 9 Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 10 Co-first author *Correspondence: zhang@broadinstitute.org http://dx.doi.org/10.1016/j.cell.2015.09.038 SUMMARY The 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 sin- gle-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 interfer- ence with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. More- over, Cpf1 cleaves DNA via a staggered DNA dou- ble-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acid- aminococcus and Lachnospiraceae , with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our under- standing of CRISPR-Cas systems and advances their genome editing applications.