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  • Brief Communication
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CRISPR–Cas12b enables efficient plant genome engineering

Nature Plants volume 6, pages 202–208 (2020)Cite this article

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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.

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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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Author notes

  1. These authors contributed equally: Meiling Ming, Qiurong Ren, Changtian Pan, Yao He, Yingxiao Zhang.

Authors and Affiliations

  1. Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA

    Meiling Ming, Changtian Pan, Yingxiao Zhang, Aimee A. Malzahn & Yiping Qi

  2. Centre of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China

    Meiling Ming & Jun Wu

  3. Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China

    Qiurong Ren, Yao He, Shishi Liu, Zhaohui Zhong, Jiaheng Wang, Xuelian Zheng & Yong Zhang

  4. Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA

    Yiping Qi

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Contributions

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.

Corresponding authors

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 Information

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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