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Engineered Cpf1 Enzymes with Altered PAM Specificities

Linyi Gao, David B.T. Cox, Winston X Yan, John Manteiga, Martin Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Feng Zhang
doi: https://doi.org/10.1101/091611
Linyi Gao
Broad Institute and MIT;
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David B.T. Cox
Broad Institute and MIT;
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Winston X Yan
Broad Institute and MIT;
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John Manteiga
Broad Institute and MIT;
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Martin Schneider
Broad Institute and MIT;
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Takashi Yamano
University of Tokyo;
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Hiroshi Nishimasu
University of Tokyo;
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Osamu Nureki
University of Tokyo;
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Feng Zhang
Broad Institute of MIT and Harvard; McGovern Institute for Brain Research at MIT
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  • For correspondence: zhang@broadinstitute.org
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Abstract

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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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 04, 2016.
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Engineered Cpf1 Enzymes with Altered PAM Specificities
Linyi Gao, David B.T. Cox, Winston X Yan, John Manteiga, Martin Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Feng Zhang
bioRxiv 091611; doi: https://doi.org/10.1101/091611
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Engineered Cpf1 Enzymes with Altered PAM Specificities
Linyi Gao, David B.T. Cox, Winston X Yan, John Manteiga, Martin Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Feng Zhang
bioRxiv 091611; doi: https://doi.org/10.1101/091611

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