CRISPR–Cas12b enables efficient plant genome engineering


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.


  1. 1.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  Google Scholar 

  2. 2.

    Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Zhang, Y., Malzahn, A., Sretenovic, S. & Qi, Y. The emerging and uncultivated potential of CRISPR technology in plant science. Nat. Plants 5, 778–791 (2019).

    Article  Google Scholar 

  4. 4.

    Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol. Cell 60, 385–397 (2015).

    CAS  Article  Google Scholar 

  5. 5.

    Teng, F. et al. Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov. 4, 63 (2018).

    Article  Google Scholar 

  6. 6.

    Strecker, J. et al. Engineering of CRISPR–Cas12b for human genome editing. Nat. Commun. 10, 212 (2019).

    CAS  Article  Google Scholar 

  7. 7.

    Yang, H., Gao, P., Rajashankar, K. R. & Patel, D. J. PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease. Cell 167, 1814–1828 (2016).

    CAS  Article  Google Scholar 

  8. 8.

    Liu, L. et al. C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism. Mol. Cell 65, 310–322 (2017).

    CAS  Article  Google Scholar 

  9. 9.

    Wu, D., Guan, X., Zhu, Y., Ren, K. & Huang, Z. Structural basis of stringent PAM recognition by CRISPR-C2c1 in complex with sgRNA. Cell Res. 27, 705–708 (2017).

    CAS  Article  Google Scholar 

  10. 10.

    Tang, X. et al. A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat. Plants 3, 17018 (2017).

    CAS  Article  Google Scholar 

  11. 11.

    Zhong, Z. et al. Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites. Mol. Plant 11, 999–1002 (2018).

    CAS  Article  Google Scholar 

  12. 12.

    Jain, I. et al. Defining the seed sequence of the Cas12b CRISPR-Cas effector complex. RNA Biol. 16, 413–422 (2019).

    Article  Google Scholar 

  13. 13.

    Paul, J. W. 3rd & Qi, Y. CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects. Plant Cell Rep. 35, 1417–1427 (2016).

    CAS  Article  Google Scholar 

  14. 14.

    Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

    CAS  Article  Google Scholar 

  15. 15.

    Lowder, L. G. et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 169, 971–985 (2015).

    Article  Google Scholar 

  16. 16.

    Lowder, L. G. et al. Robust transcriptional activation in plants using multiplexed CRISPR-Act2.0 and mTALE-act systems. Mol. Plant 11, 245–256 (2018).

    CAS  Article  Google Scholar 

  17. 17.

    Teng, F. et al. Artificial sgRNAs engineered for genome editing with new Cas12b orthologs. Cell Discov. 5, 23 (2019).

    Article  Google Scholar 

  18. 18.

    Li, Z. et al. A potent Cas9-derived gene activator for plant and mammalian cells. Nat. Plants 3, 930–936 (2017).

    CAS  Article  Google Scholar 

  19. 19.

    Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    CAS  Article  Google Scholar 

  20. 20.

    Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    CAS  Article  Google Scholar 

  21. 21.

    Tang, X. et al. A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Mol. Plant 9, 1088–1091 (2016).

    CAS  Article  Google Scholar 

  22. 22.

    You, Q. et al. CRISPRMatch: An automatic calculation and visualization tool for high-throughput CRISPR genome-editing data analysis. Int J. Biol. Sci. 14, 858–862 (2018).

    CAS  Article  Google Scholar 

  23. 23.

    Liu, W. et al. DSDecode: A web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Mol. Plant 8, 1431–1433 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

    CAS  Article  Google Scholar 

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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.

Author information




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).

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