Engineering of CRISPR-Cas12b for human genome editing

Jonathan Strecker, Sara Jones, Balwina Koopal, Jonathan Schmid-Burgk, Bernd Zetsche, Linyi Gao, Kira S. Makarova, Eugene V. Koonin and Feng Zhang ()
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Jonathan Strecker: Howard Hughes Medical Institute
Sara Jones: Howard Hughes Medical Institute
Balwina Koopal: Howard Hughes Medical Institute
Jonathan Schmid-Burgk: Howard Hughes Medical Institute
Bernd Zetsche: Howard Hughes Medical Institute
Linyi Gao: Howard Hughes Medical Institute
Kira S. Makarova: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health
Eugene V. Koonin: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health
Feng Zhang: Howard Hughes Medical Institute

Nature Communications, 2019, vol. 10, issue 1, 1-8

Abstract: Abstract The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.

Date: 2019
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DOI: 10.1038/s41467-018-08224-4

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