Engineered Cpf1 variants with altered PAM specificities View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2017-06-05

AUTHORS

Linyi Gao, David B T Cox, Winston X Yan, John C Manteiga, Martin W Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Nicola Crosetto, Feng Zhang

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. More... »

PAGES

789-792

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nbt.3900

DOI

http://dx.doi.org/10.1038/nbt.3900

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1085865597

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/28581492


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