Subburaj Saminathan, Zanatta Caroline Bedin, Nunn Jennifer A L, Hoepers Aline Martins, Nodari Rubens Onofre, Agapito-Tenfen Sarah Zanon
NORCE Norwegian Research Centre AS, Department of Climate & Environment, Tromsø, Norway.
Department of Crop Science, Federal University of Santa Catarina, Florianópolis, Brazil.
Front Plant Sci. 2022 Jul 12;13:939997. doi: 10.3389/fpls.2022.939997. eCollection 2022.
CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the () gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targeted deep sequencing strategies. We were able to identify different mutation patterns within insertions and deletions (InDels) between + 5 nt and -30 bp and mutation frequency ranging from 4.2 to 18.1% in the locus. Our results showed that DNA-free delivery of Cas9 complexes to protoplasts is a useful approach to perform early-stage genetic screens and anticipated analysis of Cas9 activity in soybeans.
基于CRISPR/Cas9的核糖核蛋白（RNP）介导系统具有将与载体DNA不必要引入和重组DNA随机整合相关的影响降至最低的特性。在此，我们描述了一个基于将Cas9核糖核蛋白直接递送至大豆原生质体的平台，用于在不转染DNA载体的情况下对基因敲除编辑的大豆品系进行遗传筛选。该平台基于大豆原生质体的分离和Cas核糖核蛋白复合物的递送。为了实证测试我们的平台，我们从大豆遗传工具箱中选择了一个模型基因。我们使用了五个不同的引导RNA（gRNA）序列，它们靶向与大豆毛状体生长相关的（）基因。此外，首次针对大豆优化了高效的原生质体转化、Cas9和gRNA的浓度及比例。使用桑格测序法和靶向深度测序策略分析了靶向诱变插入和缺失频率及序列。我们能够在+5 nt至-30 bp之间的插入和缺失（InDels）中鉴定出不同的突变模式，并且在该位点的突变频率为4.2%至18.1%。我们的结果表明，将Cas9复合物无DNA递送至原生质体是在大豆中进行早期遗传筛选以及预期分析Cas9活性的一种有用方法。
