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Efficient genome editing by CRISPR-Mb3Cas12a in mice.

ABSTRACT: As an alternative and complementary approach to Cas9-based genome editing, Cas12a has not been widely used in mammalian cells largely due to its strict requirement for the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) can efficiently edit the mouse genome based on the TTV PAM sequence with minimal numbers of large on-target deletions or insertions. When TTTV PAM sequence-targeting CRISPR (cr)RNAs of 23 nt spacers are used, >70% of the founders obtained are edited. Moreover, the use of Mb3Cas12a tagged to monomeric streptavidin (mSA) in conjunction with biotinylated DNA donor template leads to high knock-in efficiency in two-cell mouse embryos, with 40% of founders obtained containing the desired knock-in sequences.

SUBMITTER: Wang Z

PROVIDER: S-EPMC7240296 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Efficient genome editing by CRISPR-Mb3Cas12a in mice.

Wang Zhuqing Z Wang Yue Y Wang Shawn S Gorzalski Andrew J AJ McSwiggin Hayden H Yu Tian T Castaneda-Garcia Kimberly K Prince Brian B Wang Hetan H Zheng Huili H Yan Wei W

Journal of cell science 20200511 9

As an alternative and complementary approach to Cas9-based genome editing, Cas12a has not been widely used in mammalian cells largely due to its strict requirement for the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (<i>Moraxella bovoculi</i> AAX11_00205) can efficiently edit the mouse genome based on the TTV PAM sequence with minimal numbers of large on-target deletions or insertions. When TTTV PAM sequence-targeting CRISPR (cr)RNAs of 23 nt spacers are used, ...[more]

PMID: 32393674

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