Engineered Cpf1 variants with altered PAM specificities
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Author(s)
Schneider, Martin W
Yamano, Takashi
Nishimasu, Hiroshi
Nureki, Osamu
Crosetto, Nicola
Gao, Linyi
Cox, David Benjamin Turitz
Yan, Winston Xia
Manteiga, John Colonnese
Zhang, Feng
Date Issued
June 2017
Journal
Nature Biotechnology
Publisher
Nature Publishing Group
Citation
Gao, Linyi et al. “Engineered Cpf1 Variants with Altered PAM Specificities.” Nature Biotechnology 35, 8 (June 2017): 789–792 © 2017 Macmillan Publishers Limited, part of Springer Nature
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Author's final manuscript
Abstract
The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. 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 BLISS 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.
MIT Department
Institute for Medical Engineering and Science
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
McGovern Institute for Brain Research at MIT
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DOI of Published Version
https://doi.org/10.1038/nbt.3900
