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Engineered Cpf1 Enzymes with Altered PAM Specificities

By Linyi Gao, David B.T. Cox, Winston X Yan, John Manteiga, Martin Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Feng Zhang

Posted 04 Dec 2016
bioRxiv DOI: 10.1101/091611 (published DOI: 10.1038/nbt.3900)

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously, as well as low rates of off-target activity. However, the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which has been successfully harnessed for genome editing, can only robustly cleave target sites preceded by a TTTV protospacer adjacent motif (PAM), which may limit its practical utility. To address this limitation, we used a structure- guided saturation mutagenesis screen to increase the targeting range of Cpf1. We engineered two variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 to one cleavage site for every ~8.7 bp in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox.

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