A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins

초록

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. A type V CRISPR effector, LbCpf1(Cas12a) fromLachnospiraceae bacteriumND 2006, has been used for precision editing of the plant genome. We report that callus-derived protoplasts from three soybeans, includingGlycine Maxvar. 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 ofGlymaFAD2-1AandGlymaFAD2-1Btransfected 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 twoFAD2genes. 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 twoFAD2genes 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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