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bioRxiv · 10.1101/091611

Engineered Cpf1 Enzymes with Altered PAM Specificities

Gao, L.·Cox, D. B. T.·Yan, W. X.·Manteiga, J.·Schneider, M.·Yamano, T.·Nishimasu, H.·Nureki, O.·Zhang, F.
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Abstract

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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Gao, L., Cox, D. B. T., Yan, W. X., Manteiga, J., Schneider, M., Yamano, T., Nishimasu, H., Nureki, O., Zhang, F.. 2016-12-04. Engineered Cpf1 Enzymes with Altered PAM Specificities. https://doi.org/10.1101/091611

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