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  • Protocol Extension
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Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins

Nature Protocols volume 13, pages 413–430 (2018)Cite this article

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Abstract

This protocol is an extension to: Nat. Protoc. 9, 2395–2410 (2014); doi:10.1038/nprot.2014.157; published online 18 September 2014

In recent years, CRISPR/Cas9 has emerged as a powerful tool for improving crop traits. Conventional plant genome editing mainly relies on plasmid-carrying cassettes delivered by Agrobacterium or particle bombardment. Here, we describe DNA-free editing of bread wheat by delivering in vitro transcripts (IVTs) or ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 by particle bombardment. This protocol serves as an extension of our previously published protocol on genome editing in bread wheat using CRISPR/Cas9 plasmids delivered by particle bombardment. The methods we describe not only eliminate random integration of CRISPR/Cas9 into genomic DNA, but also reduce off-target effects. In this protocol extension article, we present detailed protocols for preparation of IVTs and RNPs; validation by PCR/restriction enzyme (RE) and next-generation sequencing; delivery by biolistics; and recovery of mutants and identification of mutants by pooling methods and Sanger sequencing. To use these protocols, researchers should have basic skills and experience in molecular biology and biolistic transformation. By using these protocols, plants edited without the use of any foreign DNA can be generated and identified within 9–11 weeks.

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Figure 1: Overview of CRISPR/Cas9 IVT- or RNP-mediated genome editing in wheat.
Figure 2: Schematic of PCR strategies for next-generation sequencing of bombarded immature embryos to confirm CRISPR/Cas9 delivery (Steps 38–46).
Figure 3: Anticipated results for mutagenesis by CRISPR/Cas IVTs/RNPs.

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Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0101804), the National Transgenic Science and Technology Program (2016ZX08010-002), the Chinese Academy of Sciences (QYZDY-SSW-SMC030 and GJHZ1602) and the National Natural Science Foundation of China (31788103, 31420103912 and 31570369).

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Author notes

  1. Zhen Liang and Kunling Chen: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, and Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Zhen Liang, Kunling Chen, Yi Zhang, Jinxing Liu & Caixia Gao

  2. University of Chinese Academy of Sciences, Beijing, China

    Zhen Liang & Yi Zhang

  3. Key Laboratory of Plant Stress Research, College of Life Science, Shandong Normal University, Jinan, China

    Yi Zhang

  4. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Kangquan Yin & Jin-Long Qiu

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  1. Zhen Liang
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Contributions

Z.L., K.C. and C.G. designed the experiments for the protocol; Z.L. and Y.Z. performed the experiments with assistance from K.C., J.L., K.Y. and J.-L.Q.; and Z.L., K.C. and C.G. wrote the manuscript.

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Correspondence to Caixia Gao.

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The authors declare no competing financial interests.

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Supplementary Table 1 and Supplementary Notes 1–3. (PDF 449 kb)

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Liang, Z., Chen, K., Zhang, Y. et al. Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins. Nat Protoc 13, 413–430 (2018). https://doi.org/10.1038/nprot.2017.145

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