The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1,2,3,4,5,6,7. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS7 indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Sequence Read Archive
Protein Data Bank
We thank A. Magnell for experimental assistance; R. Macrae for a critical review of the manuscript; and the entire Zhang laboratory for support and advice. D.B.T.C. is supported by T32GM007753 from the National Institute of General Medical Sciences. W.X.Y. is supported by T32GM007753 from the National Institute of General Medical Sciences and a Paul and Daisy Soros Fellowship. J.C.M. is supported by the NIH (training grant 2 T32 GM 7287-41). H.N. is supported by JST, PRESTO (JPMJPR13L8), JSPS KAKENHI (Grant Numbers 26291010 and 15H01463). O.N. is supported by the Basic Science and Platform Technology Program for Innovative Biological Medicine from the Japan Agency for Medical Research and Development, AMED, and the Council for Science, and Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology. N.C. is supported by the Karolinska Institutet, the Swedish Research Council (521-2014-2866), the Swedish Cancer Research Foundation (CAN 2015/585), and the Ragnar Söderberg Foundation. F.Z. is a New York Stem Cell Foundation–Robertson Investigator. F.Z. is supported by the NIH through NIMH (5DP1-MH100706 and 1R01-MH110049), NSF, Howard Hughes Medical Institute, the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations; and James and Patricia Poitras, Robert Metcalfe, and David Cheng.
Integrated supplementary information
Supplementary Figures 1–11 and Supplementary Tables 1–4
About this article
Nature Communications (2018)