利用CRISPR/Cas13技术抑制SPCSV-RNase3介导的病毒协生作用及改良甘薯病毒病抗性的研究

Sweet potato virus disease (SPVD), which is caused by the co-infection of two RNA viruses (sweet potato chlorotic stunt virus: SPCSV and sweet potato feathery mottle virus: SPFMV), has great impact on the sweet potato industry in China and even all over the world. However, there is no suitable genetic improvement method to enhance SPVD resistance. Cas13 is a newly-discovered type VI CRISPR (Clustered regularly interspaced short palindromic repeats)-associated RNA-guided ribonuclease that cleaves RNA with high efficiency and specificity. This provides an improved tool for cleaving viral RNAs and protecting plants from the infection of RNA viruses. Therefore, CRISPR/Cas13 technique has great application in the prevention and control of plant RNA viruses. In this project, we intend to use CRISPR/Cas13 system to enhance SPVD resistance and this is mainly achieved by targeting SPCSV-RNase3, which is a viral RNA silencing suppressor as well as core pathogenic factor causing viral synergism. Firstly, we plan to use Nicotiana benthamiana as transient experimental system to screen efficient Cas13 enzymes and guide RNAs (gRNAs) and to evaluate the effect of transient RNase3-cleaving on the viral synergism. Furthermore, we intend to generate Cas13-gRNAs transgenic sweet potato by using of Agrobacterium tumefaciens-mediated transformation method. Subsequently, the SPVD resistance of Cas13-gRNAs transgenic sweet potato will be investigated comprehensively. The output of this project will provide new idea, solid theoretical basis and reliable technical system for the genetic improvement of SPVD resistance.

甘薯褪绿矮化病毒(SPCSV)和甘薯羽状斑驳病毒(SPFMV)协生共侵染引起的甘薯病毒病(SPVD)严重危害我国乃至世界甘薯产业。目前尚无合适的遗传改良方法用于提高SPVD抗性。CRISPR/Cas13技术能够直接靶向干涉RNA，在防治植物RNA病毒病中具有较大应用前景。本项目拟利用该技术靶向干涉SPCSV-RNase3 (RNA沉默抑制子，SPVD致病关键因子)，希望通过抑制其介导的病毒协生作用来改良SPVD抗性。本项目首先以本氏烟作为研究材料，利用瞬时CRISPR/Cas13实验技术体系筛选有效Cas13-gRNAs组合, 并研究RNase3瞬时靶向干涉抑制病毒协生作用的效果；之后，通过遗传转化方式获得Cas13-gRNAs转基因甘薯并全面分析其SPVD抗性，最终获得SPVD抗性改良的甘薯新材料。本项目研究成果可为SPVD抗性遗传改良提供全新的思路方法、坚实的理论依据和可靠的技术体系。

甘薯是保障我国粮食安全的底线作物，近年来甘薯病毒病（SPVD)的流行严重危害我国甘薯产业健康发展。SPVD由甘薯褪绿矮化病毒(SPCSV)和甘薯羽状斑驳病毒(SPFMV)协生共侵染所引起，目前尚无有效的SPVD抗性遗传改良技术。本项目针对SPVD致病关键因子SPCSV-RNase3, 提出利用CRISPR-Cas13系统靶向RNase3进行SPVD的抗性遗传改良，获得以下重要结果：（1）开发了基于甘薯双生病毒（SPLCV）复制元件的高效CRISPR-Cas表达载体，显著提高了LwaCas13a介导的RNA编辑效率和SpCas9/LbCas12a介导的基因编辑效率；（2）在本氏烟中建立了Cas13变体RNA靶向效率比较的技术体系，系统比较了4种Cas13(LshCas13a、LwaCas13a、PspCas13b和RfxCas13d)的外源RNA靶向效率和RNA病毒靶向效率，发现LwaCas13a和RfxCas13d具有较高RNA靶向效率；（3）建立和优化了RNase3的异源病毒表达体系（CMV-RNase3）并建立了RNase3介导的病毒协生体系，实现利用本氏烟进行SPCSV-RNase3相关生物学功能的研究；（4）利用本氏烟实验系统，证明靶向RNase3的LwaCas13a能够显著降低RNase3对寄主RNAi活性的抑制作用，同时证明能够提高本氏烟对RNase3介导的病毒协生效应的抗性；（5）设计并优化了靶向SPCSV-RNase3的RfxCas13d载体，转入甘薯（徐薯29）胚性愈伤细胞并获得了2个转化阳性材料（RfxCas13d空载和RfxCas13d靶标载体），通过嫁接接种病毒和自然虫媒接种病毒，证明RfxCas13d靶标载体转化植株的SPVD抗性显著提升。本课题研究结果为SPVD抗性遗传改良提供了重要理论基础与技术支撑，创制出的SPVD抗性种质为我国甘薯产业提供了特异种质资源，今后或将有重要应用和推广价值。同时，本项目所开发的SPLCV高效CRISPR-Cas递送载体也将在甘薯基因编辑育种中有着重要的应用。