内含肽介导的撕裂Cas9:一种非转基因的植物基因编辑系统

Intein-Mediated Split-Cas9:a Transgene-Free Plant Genome Editing System

CRISPR/Cas9技术在植物的基础研究以及谷物的遗传改造方面展现出了前所未有的潜力.目前植物基因编辑方法大多是基于农杆菌介导的遗传转化,该方法不仅涉及转基因过程,容易引发公众关注,同时该方法对较难转化的植物而言有一定的技术挑战.研究开发了一个简单的非转基因植物基因编辑方法,即采用病毒递送的方式将片段化的Cas9和gRNA运送到烟草中.为了适应马铃薯病毒X载体的运载能力,将金黄色葡萄球菌SaCas9分成3个部分,用2个片段化的内含肽将其连接成完整的Cas9蛋白.结果表明:在培养的细胞和植物叶片中,2个片段化的内含肽均能使3个片段化的Cas9重组为完整的Cas9蛋白.将3个片段化的载体和1个sgRNA载体注射到叶片中,能对PDS基因进行靶向编辑.该非转基因的植物基因编辑方法可能对其他多种植物有一定的应用效果.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)-induced genomic engineering has shown unprecedented perspectives for basic plant research and crop genetic improvements.However,current plant genome editing methods are mostly based on Agrobacterium transformation.This procedure involves transgenic intermediates that could raise regulatory concernsand technical challenges when used with hard-to-transform plants.Here,we describe a simple transgene-free plant editing method for tobacco using viral delivery of spliced Cas9/gRNA.To fit the delivery capacity of the systemic infectious potato virus X vector,we split the Staphylococcus aureus Cas9 into three parts by combining two split-inteins.We showed that trans-splicing two inteins reconstitutes the full Cas9 protein in cultured cells and plant leaves.The direct injection of three split-Cas9 vectors and a gRNA vector into leaves could induce targeted genomic modifications with high efficiency.Our method revealed that Cas9 could be trans-spliced into three separate parts.This new simple transgene-free plant editing method may be applicable to various other plant species.