新疆农大刘晓东团队利用FT移动基因引导RNA和DNA病毒进行可遗传基因编辑

来源：植物生物学

病毒诱导的基因组编辑(VIGE)系统可用于快速鉴定基因功能和生成基因敲除文库，以替代病毒诱导的基因沉默(VIGS)。

尽管植物病毒介导的VIGE有很大的应用前景，但由于病毒不能进入植物的茎尖分生组织(SAM)，因此利用该系统无法将编辑好的基因转移到下一代。

结果：我们构建了一种新型的棉花皱叶病毒(CLCrV)介导的VIGE系统，通过转化Cas9过表达(Cas9-OE)拟南芥，将BRI1、GL2、PDS和GUS基因导入拟南芥。针对VIGE系统的缺陷，将102bp的FT mRNAs与sgRNAs融合，转化Pro姚：：Cas9和Pro35S：：Cas9，以探讨开花位点T(FT)基因在将sgRNAs导入SAM中的功能，从而避免组织培养，稳定获得春季可遗传突变体。

结果表明，sgRNAs与FT mRNA在5‘端的融合(FT策略)能有效地使受感染植株进行基因编辑，并能获得下一代可遗传的突变，其效率为4.35-8.79%。此外，经FT-sgRNAs编辑的春季病毒不含CLCrV基因组的任何成分。

结论：基于CLCrV介导的VIGE系统，利用FT策略可以获得遗传突变后代，避免了组织培养和稳定转化。

Background: The virus-induced genome editing (VIGE) system can be used to quickly identify gene functions and generate knock-out libraries as an alternative to the virus-induced gene silencing (VIGS). Although plant virusmediated VIGE has been shown to have great application prospects, edited genes cannot be transferred to the next generations using this system, as viruses cannot enter into shoot apical meristem (SAM) in plants. Results: We developed a novel cotton leaf crumple virus (CLCrV)-mediated VIGE system designed to target BRI1, GL2, PDS genes, and GUS transgene in A. thaliana by transforming Cas9 overexpression (Cas9-OE) A. thaliana. Given the defciency of the VIGE system, ProYao::Cas9 and Pro35S::Cas9 A. thaliana were transformed by fusing 102 bp FT mRNAs with sgRNAs so as to explore the function of Flowering Locus T (FT) gene in delivering sgRNAs into SAM, thus avoiding tissue culture and stably acquiring heritable mutant ofspring. Our results showed that sgRNAs fused with FT mRNA at the 5′ end (FT strategy) efectively enabled gene editing in infected plants and allowed the acquisition of mutations heritable by the next generation, with an efciency of 4.35–8.79%. In addition, gene-edited ofspring by FT-sgRNAs did not contain any components of the CLCrV genome. Conclusions: FT strategy can be used to acquire heritable mutant offspring avoiding tissue culture and stable transformation based on the CLCrV-mediated VIGE system in A. thaliana. 

Keywords: CLCrV, CRISPR/Cas9, FT-sgRNA, VIGE