CRISPR–Cas12b enables efficient plant genome engineering

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12b is a newly emerged genome engineering system. Here, we compared Cas12b from Alicyclobacillus acidoterrestris (Aac), Alicyclobacillus acidiphilus (Aa), Bacillus thermoamylovorans (Bth) and Bacillus hisashii (Bh) for genome engineering in rice, an important crop. We found AaCas12b was more efficient than AacCas12b and BthCas12b for targeted mutagenesis, which was further demonstrated in multiplexed genome editing. We also engineered the Cas12b systems for targeted transcriptional repression and activation. Our work establishes Cas12b as the third promising CRISPR system, after Cas9 and Cas12a, for plant genome engineering.

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Fig. 1: Comprehensive analysis of three CRISPR–Cas12b systems for genome editing in rice protoplasts.
Fig. 2: Singular and multiplexed gene editing in rice T0 lines by AacCas12b and AaCas12b.
Fig. 3: Effective CRISPR interference and CRISPR activation by dCas12b systems.

Data availability

The 29 Gateway compatible vectors for the CRISPR–Cas12b systems are available from Addgene: pYPQ290 (no. 129670), pYPQ291 (no. 129671), pYPQ292 (no. 129672), pYPQ290-D570A (no. 129673), pYPQ290-D977A (no. 129674), pYPQ290-E848A (no. 129675), pYPQ291-D573A (no. 129676), pYPQ291-D951A (no. 129677), pYPQ291-E827A (no. 129678), pYPQ292-D570A (no. 129679), pYPQ292-D977A (no. 129680), pYPQ292-E848A (no. 129681), pYPQ290-D570A-SRDX (no. 129682), pYPQ291-D573A-SRDX (no. 129683), pYPQ292-D570A-SRDX (no. 129684), pYPQ141-ZmUbi-RZ-Aac (no. 129685), pYPQ141-ZmUbi-RZ-Bth (no. 129686), pYPQ141-ZmUbi-RZ-Aa1.2.3 (no. 136372), pYPQ141-ZmUbi-RZ-Aa1.2 (no. 136373), pYPQ141-ZmUbi-RZ-Aa3.8.3 (no. 136374), pYPQ141-ZmUbi-RZ-Aa3.8.4 (no. 136375), pYPQ141-ZmUbi-RZ-Aa3.8 (no. 136376), pYPQ141-ZmUbi-RZ-Aac.3 (no. 136377), pYPQ141-ZmUbi-RZ-Bh (no. 136378), pYPQ239A (dFnCas12a)-TV (no. 136379), pYPQ292 (AaCas12b)-D570-TV (no. 136380), pYPQ292 (AaCas12b)-D570-TV-MS2-TV (no. 136381), pYPQ292 (AaCas12b)-D570-TV-MS2-VPR (no. 136382) and pYPQ293 (BhCas12b_v4) (no. 136383). The high-throughput sequencing data sets have been submitted to the National Center for Biotechnology information (NCBI) database under Sequence Read Archive (SRA) BioProject ID PRJNA553352.

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Acknowledgements

This work was supported by University of Maryland startup funds, the National Science Foundation Plant Genome Research Program grant (award no. IOS-1758745), the Biotechnology Risk Assessment Grant Program competitive grant (award no. 2018-33522-28789) from the US Department of Agriculture, Foundation for Food and Agriculture Research grant (award no. 593603) and Syngenta Biotechnology to Y.Q. It was also supported by the National Transgenic Major Project (award nos. 2019ZX08010003-001-002 and 2018ZX08020-003), the National Science Foundation of China (award no. 31771486), the Sichuan Youth Science and Technology Foundation (award no. 2017JQ0005) and the Science Strength Promotion Program of UESTC to Yong Z. M.M was supported by a scholarship from China Scholarship Council. A.M was supported by a scholarship from Cosmos Club Foundation.

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Y.Q. and Yong Z. designed the experiments. M.M, Yingxiao Z. and C.P. generated all the constructs. M.M., Q.R., Y.H., S.L., Z.Z., J.W. and X.Z. performed the transient assays in protoplasts. Q.R. and Y.H. prepared samples for high-throughput sequencing. M.M. generated stable transgenic rice and analysed the plants. C.P. conducted transcriptional repression and activation assays. Y.Q., Yong Z., A.M. and J.W. wrote the paper with input from other authors. All authors read and approved the final manuscript.

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Correspondence to Yong Zhang or Yiping Qi.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Sang Gyu Kim and Huawei Zhang and the other, anonymous, reviewer for their contribution to the peer review of this work.

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Supplementary methods, Supplementary Figs. 1–20 and Supplementary Tables 1 and 2.

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Ming, M., Ren, Q., Pan, C. et al. CRISPR–Cas12b enables efficient plant genome engineering. Nat. Plants 6, 202–208 (2020). https://doi.org/10.1038/s41477-020-0614-6

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