A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins
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ORIGINAL ARTICLE
A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins Hyeran Kim1 · Jisun Choi1 Received: 21 April 2020 / Accepted: 7 September 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
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. Keywords CRISPR/LbCpf1 · Soybean genome editing · Glycine Max var. Williams 82 · Glycine Max cv. Kwangan · Glycine Max cv. Daewon · Soybean callus protoplasts
Introduction The CRISPR derived from Prevotella and Francisella1 (Cpf1) is a specific genome-editing tool acting as a single RNA-guided molecular scissor and designed to induce a mutation via a staggered double-stranded break in the DNA at the target locus in any sequenced genome (Zetsche et al. Communicated by Neal Stewart. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00299-020-02597-x) contains supplementary material, which is available to authorized users. * Hyeran Kim [email protected] 1
Department of Biological Sciences, Kangwon National University, Kangwondaehak‑gil 1, Chuncheon 24341, South Korea
2015). We obtained two representative Cpf1-based CRISPR tools from different strains, such as Acidaminococcus sp. BV3L6 (As) and Lachnospiraceae bacterium ND2006 (Lb), which are robust genome-editing tools for applications involving human and plant cells (Zetsche et al. 2015; Hur et al. 2016; Kim et
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