Murovec Jana, Guček Katja, Bohanec Borut, Avbelj Monika, Jerala Roman
Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia.
Front Plant Sci. 2018 Nov 5;9:1594. doi: 10.3389/fpls.2018.01594. eCollection 2018.
The CRISPR/Cas9 genome editing system has already proved its efficiency, versatility and simplicity in numerous applications in human, animal, microbe and plant cells. Together with the vast amount of genome and transcriptome databases available, it represents an enormous potential for plant breeding and research. Although most changes produced with CRISPR/Cas9 do not differ from naturally occurring mutations, the use of transgenesis during varietal development can still trigger GMO legislation in countries that rely on process-based regulation. Moreover, stable integration of DNA coding for genome-editing tools into plant genomes can result in insertional mutagenesis, while its prolonged expression can cause mutations in off-target sites. These pitfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and -transcribed or synthesized sgRNA. We therefore aimed to develop a DNA-free protocol for site-directed mutagenesis of three species of the genus (, and ) with the use of RNPs. We chose cabbage, rapeseed and Chinese cabbage as species representatives and introduced RNPs into their protoplasts with PEG 4000. Four sgRNAs targeting two endogenous genes (the and genes, two sgRNAs per gene) were introduced into all three species. No mutations were detected after transfection of rapeseed protoplasts, while we obtained mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in cabbage and Chinese cabbage, respectively. In both species, a positive correlation was displayed between the amount (7.5, 15, 30, and 60 μg) of Cas9 enzyme and sgRNA introduced and mutation frequency. Nucleotide changes (insertions and deletions) were detected 24 h after transfection and did not differ 72 h after transfection. They were species-, gene- and locus-dependent. In summary, we demonstrated the suitability of RNP transfection into and protoplasts for high-efficiency indel induction of two endogenous genes. Due to the relatively high mutation frequencies detected (up to 24.51%), this study paves the way for regeneration of precisely mutated plants without the use of transgenesis.
CRISPR/Cas9基因组编辑系统已在人类、动物、微生物和植物细胞的众多应用中证明了其高效性、多功能性和简易性。连同现有的大量基因组和转录组数据库，它在植物育种和研究方面展现出巨大潜力。尽管CRISPR/Cas9产生的大多数变化与自然发生的突变并无不同，但在品种培育过程中使用转基因技术仍可能在依赖基于过程监管的国家触发转基因法规。此外，编码基因组编辑工具的DNA稳定整合到植物基因组中可能导致插入诱变，而其长时间表达可能会在脱靶位点引起突变。通过递送由纯化的重组酶Cas9和转录或合成的sgRNA组成的预组装核糖核蛋白复合物（RNP），可以避免这些问题。因此，我们旨在开发一种不使用DNA的方案，利用RNP对芸苔属的三个物种（甘蓝、油菜和大白菜）进行定点诱变。我们选择甘蓝、油菜和大白菜作为物种代表，并通过聚乙二醇4000将RNP导入它们的原生质体。针对两个内源基因（BnALS和BraA.PDS基因，每个基因两个sgRNA）的四个sgRNA被导入所有三个物种。油菜原生质体转染后未检测到突变，而在甘蓝和大白菜中我们分别获得了0.09%至2.25%和1.15%至24.51%的突变频率。在这两个物种中，导入的Cas9酶和sgRNA的量（7.5、15、30和60μg）与突变频率之间呈现正相关。转染后24小时检测到核苷酸变化（插入和缺失），转染后72小时无差异。它们具有物种、基因和位点依赖性。总之，我们证明了将RNP转染到甘蓝和大白菜原生质体中对于高效诱导两个内源基因的插入缺失是合适的。由于检测到相对较高的突变频率（高达24.51%），本研究为不使用转基因技术再生精确突变的芸苔属植物铺平了道路。
