解屹 徐翔 葛明 张万红 黄坤 江连强 赵世民 王玉洁 彭子忠 廖成 杨金广 王凤龙

摘 要：CRISPR-Cas适应性免疫系统为细菌和古细菌免受外来核酸和噬菌体的侵害提供了保护。其中2类VI-A型CRISPR-Cas效应子Cas13a（之前称为C2c2），可受CRISPR RNA的引导，靶向切割与原始间隔区反向互补的单链RNA。本研究利用CRISPR-Lsh-Cas13a系统，多靶标编辑烟草花叶病毒（TMV）RdRP、MP和CP基因，以抑制病毒在寄主烟草体内的侵染与扩散，从而达到保护寄主的作用。该系统在瞬时表达测定中表现出对野生型TMV-U1和表达绿色荧光蛋白的TMV-30b的干扰以及对病毒累积和病毒病症状的抑制。靶向TMV RdRP基因的CRISPR RNA表现出比靶向MP和CP基因更好的编辑效能。经过适当设计的CRISPR-Lsh-Cas13a系统被赋予了广谱抗性，可以特异性编辑多种TMV菌株。研究表明，CRISPR-Cas13a系统可以用于针对RNA病毒的抑制干扰。

关键词：基因编辑;CRISPR-Cas13a;烟草花叶病毒;病毒防控;多靶标编辑

Abstract： The CRISPR-Cas adaptive immune system provides protection for bacteria and archaea from foreign nucleic acids and bacteriophages. In the CRISPR-Cas adaptive immune system， two classes of the VI-A CRISPR-Cas effector Cas13a （previously called C2c2） can be guided by CRISPR RNA to target and then cut the single-stranded RNA that is reversely complementary to the protospacers. In this study， the CRISPR-Lsh-Cas13a system was used for the multi-target editing of RdRP， MP， and CP genes of Tobacco mosaic virus （TMV） to protect the host from infection and spread of the virus. This system showed interference with wild-type TMV-U1 and TMV-30b expressing green fluorescent proteins in transient expression assays， as well as suppression of virus accumulation and viral disease symptoms. The CRISPR RNA targeting TMV RdRP gene showed better editing efficiency than that targeting MP and CP genes. The appropriately designed CRISPR-Lsh-Cas13a system was endowed with broad-spectrum resistance and can specifically edit a variety of TMV strains. This study showed that the CRISPR-Cas13a system can be used for the suppression and interference against RNA viruses.

Keywords： gene editing; CRISPR-Cas13a; tobacco mosaic virus; virus prevention and control; multi-target editing

植物RNA病毒种类繁多，其中最具代表性的烟草花叶病毒TMV（Tobacco mosaic virus）富有极强的侵染性、抗逆性和危害性，寄主范围十分广泛，可侵染150余属350余种植物[1]，包括烟草、番茄、马铃薯、菠菜等多种作物。一旦发病会造成大规模扩散感染，导致作物产量、品质严重下降，从而造成严重的经济损失[2]。据报道，世界各地每年因TMV侵染作物造成的经济损失超过1亿美元[3]。TMV可从微伤口处侵染寄主。发病初期，寄主新叶叶脉及周围叶肉组织出现半透明明脉症状;进而病毒持续复制增殖，严重干扰寄主自身细胞分裂，导致叶片厚薄不均、畸形、皱缩，并呈现典型的花叶症状，植株也随之生长迟缓，呈现矮化、节间缩短等症状，侵染严重的植株逐渐坏死凋亡。……