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Efficient genome editing in wheat using Cas9 and Cpf1 (AsCpf1 and LbCpf1) nucleases

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Abstract

Genome editing can be used to create new wheat varieties with enhanced performance. Clustered regularly interspaced short palindromic repeat (CRISPR) is a powerful tool for knockout generation, precise modification, multiplex engineering, and the activation and repression of target genes. Targeted mutagenesis via RNA-guided genome editing using type II CRISPR-Cas9 is highly efficient in some plant species, but not in others. One possible solution is to use newly discovered variants of genome editing enzymes such as the class 2 system component Cpf1 (CRISPR from Prevotella and Francisella 1) in place of the more commonly used Cas9. We compared the editing efficiency of Cas9 and two Cpf1 orthologs, AsCpf1 (Acidaminococcus spp. BV3L6) and LbCpf1 (Lachnospiraceae bacterium ND2006) in wheat (Triticum aestivum). LbCpf1 had a higher editing efficiency for the target gene TaPDS than AsCpf1 and Cas9, and Cas9 induced more off-target mutations than AsCpf1 and LbCpf1, suggesting that CRISPR-LbCpf1 is a powerful genome editing tool for polyploid plants such as wheat.

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Funding

This work was supported by the Montana Wheat and Barley Committee Grant and Winifred-Asbjornson Plant Science Endowment.

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  1. Dongjin Kim and Megan Hager contributed equally to this work.

Authors and Affiliations

  1. Cereal Genomics Lab, Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, USA

    Dongjin Kim & Megan Hager

  2. Department of Agronomy, University of Florida, Gainsville, FL, USA

    Eleanor Brant

  3. Montana BioAg. Inc., Missoula, MT, USA

    Hikmet Budak

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  1. Dongjin Kim
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  2. Megan Hager
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  4. Hikmet Budak
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HB conceived and designed this study. DK, MH, and EB performed the experiments, and DK, MH, EB, and HB wrote and edited the manuscript and generated the figures. All authors read and approved the manuscript.

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Correspondence to Hikmet Budak.

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Kim, D., Hager, M., Brant, E. et al. Efficient genome editing in wheat using Cas9 and Cpf1 (AsCpf1 and LbCpf1) nucleases. Funct Integr Genomics 21, 355–366 (2021). https://doi.org/10.1007/s10142-021-00782-z

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  • DOI: https://doi.org/10.1007/s10142-021-00782-z

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