Abstract
Key message
Calli protoplasts isolated from three soybean cultivars are useful tools to evaluate guide RNAs for clustered regularly interspaced short palindromic repeats (CRISPR)-based precise gene editing.
Abstract
A type V CRISPR effector, LbCpf1(Cas12a) from Lachnospiraceae bacterium ND 2006, has been used for precision editing of the plant genome. We report that callus-derived protoplasts from three soybeans, including Glycine Max var. Williams 82 and two Korean cultivars (Kwangan and Daewon) represent efficient systems for the screening of active crRNA for CRISPR/LbCpf1. CRISPR/LbCpf1 ribonucleoproteins (RNPs) were delivered as complexes of purified endonucleases mixed with designed crRNA to simultaneously edit target genes of GlymaFAD2-1A and GlymaFAD2-1B transfected into three soybean protoplasts including genome-sequenced Williams 82 with cultivars, Kwangan and Daewon. Previously, we reported that nine crRNAs designed for LbCpf1 exhibited varying degrees of editing efficacy for two FAD2 genes. Among the nine crRNAs, the LbCpf1-crRNA3 complexes showed the highest efficiency in soybean cotyledon protoplasts. The new screening systems of callus protoplasts from three soybeans have been successfully used to transfect GFP-tagged markers and CRISPR/LbCpf1 RNPs. The callus protoplasts confirm that the LbCpf1-crRNA3 complex is an active crRNA for LbCpf1 to edit two FAD2 genes similar to cotyledon protoplasts. These results demonstrate that soybean callus protoplast-based CRISPR/crRNA selection is a new and practical tool to screen the efficacy of crRNAs and a prerequisite for progressive regeneration of the edited soybean.
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Acknowledgements
We thank Prof. Ik-Young Choi, Prof. Jeong Dong Lee, and Dr. Jung-Kyung Moon for providing initial soybean materials, and Ms. Hyunah Lee in the Central Laboratory of Kangwon National University for confocal microscopy (LSM880 with Airyscan). We deeply appreciate the technical contributions provided by Ms. Min Kyung Choi and Dr. Jiyeon Kweon. Both authors appreciate PCGE lab members.
Funding
This work was supported by the Basic Science Research Program of National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology [Grant No.2018R1A2B6006233], and the New Breeding Technologies Development Program, Rural Development Administration (RDA), Republic of Korea [Grant No. PJ01477602] to HK and The National Research Foundation of Korea and the Center for Women in Science, Engineering, and Technology [Grant No. WISET-2019-674ho] to J.C.
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HK designed and performed the project. JC and HK performed and assisted experiments. HK wrote the manuscript. HK supervised the project. Both authors approved the manuscript.
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Communicated by Neal Stewart.
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Kim, H., Choi, J. A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins. Plant Cell Rep 40, 1059–1070 (2021). https://doi.org/10.1007/s00299-020-02597-x
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DOI: https://doi.org/10.1007/s00299-020-02597-x