Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.
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We would like to thank F.A. Ran for helpful discussions and overall support, and B. Cartigny and J. van den Bogaerde for technical assistance, and the entire Zhang laboratory for support and advice. 6-His-MBP-TEV, a pET-based vector, was kindly given to us by Doug Daniels of the Broad Institute. M.H. was supported by the Human Frontiers Scientific Program. O.A.A. is supported by a Paul and Daisy Soros Fellowship and a Friends of the McGovern Institute Fellowship. J.S.G. is supported by a D.O.E. Computational Science Graduate Fellowship. E.M.D.G. is supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB), of the National Institutes of Health (5T32EB1680). K.S. is supported by an NIH grant GM10407, Russian Science Foundation grant 14-14-00988, and Skoltech. J.v.d.O. is supported by Netherlands Organization for Scientific Research (NWO) through a TOP grant (714.015.001). F.Z. is supported by the NIH through NIMH (5DP1-MH100706 and 1R01-MH110049); by the New York Stem Cell, Poitras, Simons, Paul G. Allen Family, and Vallee Foundations; and by David R. Cheng, Tom Harriman, and B. Metcalfe. F.Z. is a New York Stem Cell Foundation Robertson Investigator. The authors plan to make the reagents widely available to the academic community through Addgene and to provide software tools via the Zhang lab website (http://www.genome-engineering.org/).
A patent has been filed relating to the presented data. F.Z. is a founder and scientific advisor for Editas Medicine, and a scientific advisor for Horizon Discovery.
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Zetsche, B., Heidenreich, M., Mohanraju, P. et al. Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Nat Biotechnol 35, 31–34 (2017). https://doi.org/10.1038/nbt.3737
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