Abstract

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.

中文翻译：

---

植物中马铃薯病毒X载体介导的无DNA基因组编辑

摘要

基因组编辑技术对于植物科学和作物育种很重要。使用一般的CRISPR-Cas9方法制备的基因组编辑的植物通常含有外源DNA，这对于生产用于营养繁殖或高度杂合的杂交品种的无基因组编辑的无转基因植物来说是有问题的。在这里，我们描述了一种通过使用马铃薯X病毒（PVX）载体表达Cas9和单向导（sg）RNA来在本氏烟草中高效靶向诱变的方法。继农杆菌介导的病毒载体cDNA导入，> 60％的不经抗生素选择就再生的芽带有目标突变，而≤18％的芽中含有T-DNA。PVX载体还用于表达碱基编辑器，该碱基编辑器由修饰的Cas9与胞嘧啶脱氨酶融合而成，可在再生芽中引入靶向核苷酸取代。我们还报告了通过机械接种包含表达Cas9的PVX载体的病毒粒子的外源无DNA基因组编辑。这种简单有效的病毒载体介导的CRISPR-Cas9传递可促进植物中无转基因的基因编辑。