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Plant Gene Knockout and Knockdown by CRISPR-Cpf1 (Cas12a) Systems

  • Yingxiao Zhang
  • Yong Zhang
  • Yiping QiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

Abstract

CRISPR-Cpf1 (Cas12a) is a class II type V endonuclease, which has been used as a genome editing tool in different biological systems. Here we describe a fast, efficient, and user-friendly system for CRISPR-Cpf1 expression vector assembly. In this system, the Pol II promoter is used to drive the expression of both Cpf1 and its crRNA, with the crRNA flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozyme RNAs for precise crRNA processing. All the components of this system can be modified depending on plant species and experimental goals. Using this system, nearly 100% editing efficiency and 90% gene expression decrease were achieved in rice and Arabidopsis, respectively.

Key words

CRISPR-Cpf1 (Cas12a) Plant gene knockout Plant gene knockdown Gateway cloning 

Notes

Acknowledgements

This work is supported by funds from University of Maryland and Syngenta Biotechnology.

References

  1. 1.
    Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Endo A, Masafumi M, Kaya H, Toki S (2016) Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Sci Rep 6:38169. https://doi.org/10.1038/srep38169CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Tang X, Lowder LG, Zhang T, Malzahn AA, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y (2017) A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat Plants 3:17018. https://doi.org/10.1038/nplants.2017.103CrossRefPubMedGoogle Scholar
  4. 4.
    Xu R, Qin R, Li H, Li D, Li L, Wei P, Yang J (2017) Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnol J 15:713. https://doi.org/10.1111/pbi.12669CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Zhong Z, Zhang Y, You Q, Tang X, Ren Q, Liu S, Yang L, Wang Y, Liu X, Liu B, Zhang T, Zheng X, Le Y, Zhang Y, Qi Y (2018) Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites. Mol Plant 11:999. https://doi.org/10.1016/j.molp.2018.03.008CrossRefPubMedGoogle Scholar
  6. 6.
    Yamano T, Nishimasu H, Zetsche B, Hirano H, Slaymaker IM, Li Y, Fedorova I, Nakane T, Makarova KS, Koonin EV, Ishitani R, Zhang F, Nureki O (2016) Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165:949–962. https://doi.org/10.1016/j.cell.2016.04.003CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Zhang X, Wang J, Cheng Q, Zheng X, Zhao G, Wang J (2017) Multiplex gene regulation by CRISPR-ddCpf1. Cell Discov 3:17018. https://doi.org/10.1038/celldisc.2017.18CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Tak YE, Kleinstiver BP, Nuñez JK, Hsu JY, Horng JE, Gong J, Weissman JS, Joung JK (2017) Inducible and multiplex gene regulation using CRISPR–Cpf1-based transcription factors. Nat Methods 14:1163–1166. https://doi.org/10.1038/nmeth.4483CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Zhang X, Wang W, Shan L, Han L, Ma S, Zhang Y, Hao B, Lin Y, Rong Z (2018) Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9(4):380–383. https://doi.org/10.1007/s13238-017-0491-6CrossRefPubMedGoogle Scholar
  10. 10.
    Yin X, Biswal AK, Dionora J, Perdigon KM, Balahadia CP, Mazumdar S, Chater C, Lin H-C, Coe RA, Kretzschmar T, Gray JE, Quick PW, Bandyopadhyay A (2017) CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice. Plant Cell Rep 36:745–757. https://doi.org/10.1007/s00299-017-2118-zCrossRefPubMedGoogle Scholar
  11. 11.
    Wang M, Mao Y, Lu Y, Tao X, Zhu J (2017) Multiplex gene editing in rice using the CRISPR-Cpf1 system. Mol Plant 10:1011–1013. https://doi.org/10.1016/j.molp.2017.03.001CrossRefGoogle Scholar
  12. 12.
    Begemann MB, Gray BN, January E, Gordon GC, He Y, Liu H, Wu X, Brutnell TP, Mockler TC, Oufattole M (2017) Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases. Sci Rep 7:11606. https://doi.org/10.1038/s41598-017-11760-6CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Hu X, Wang C, Liu Q, Fu Y, Wang K (2017) Targeted mutagenesis in rice using CRISPR-Cpf1 system. J Genet Genomics 44:71–73. https://doi.org/10.1016/j.jgg.2016.12.001CrossRefPubMedGoogle Scholar
  14. 14.
    Kim H, Kim S-T, Ryu J, Kang B-C, Kim J-S, Kim S-G (2017) CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat Commun 8:14406. https://doi.org/10.1038/ncomms14406CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Liu H, Ding Y, Zhou Y, Jin W, Xie K, Chen L (2017) CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants. Mol Plant 10:530–532. https://doi.org/10.1016/j.molp.2017.01.003CrossRefPubMedGoogle Scholar
  16. 16.
    Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123. https://doi.org/10.1093/bioinformatics/btu743CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Plant Science and Landscape ArchitectureUniversity of MarylandCollege ParkUSA
  2. 2.Department of Biotechnology, School of Life Science and Technology, Center for Informational BiologyUniversity of Electronic Science and Technology of ChinaChengduChina
  3. 3.Institute for Bioscience and Biotechnology ResearchUniversity of MarylandRockvilleUSA

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