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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
1Broad Institute of MIT and Harvard Cambridge, Massachusetts 02142, USA
2Department of Biological Engineering, Cambridge, MA 02139, USA
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David B.T. Cox
1Broad Institute of MIT and Harvard Cambridge, Massachusetts 02142, USA
3Department of Biology Massachusetts Institute of Technology Cambridge, MA 02139, USA
4Harvard-MIT Division of Health Sciences and Technology, Boston, MA 02115, USA
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Winston X. Yan
1Broad Institute of MIT and Harvard Cambridge, Massachusetts 02142, USA
4Harvard-MIT Division of Health Sciences and Technology, Boston, MA 02115, USA
5Graduate Program in Biophysics Harvard Medical School Boston, MA 02115, USA
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John Manteiga
3Department of Biology Massachusetts Institute of Technology Cambridge, MA 02139, USA
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Martin Schneider
1Broad Institute of MIT and Harvard Cambridge, Massachusetts 02142, USA
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Takashi Yamano
6Department of Biological Sciences, Graduate School of Science The University of Tokyo, Tokyo 113-0032, Japan
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Hiroshi Nishimasu
6Department of Biological Sciences, Graduate School of Science The University of Tokyo, Tokyo 113-0032, Japan
7JST, PRESTO, Tokyo 113-0032, Japan
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Osamu Nureki
6Department of Biological Sciences, Graduate School of Science The University of Tokyo, Tokyo 113-0032, Japan
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Feng Zhang
1Broad Institute of MIT and Harvard Cambridge, Massachusetts 02142, USA
2Department of Biological Engineering, Cambridge, MA 02139, USA
8McGovern Institute for Brain Research, Cambridge, MA 02139, USA
9Department of Brain and Cognitive Sciences Massachusetts Institute of Technology Cambridge, Cambridge, MA 02139, USA
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  • For correspondence: zhang@broadinstitute.org
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Summary

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1-5. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously3, as well as low rates of off-target activity4, 5. 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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