Construction of Multi-target Editing Technology Based on CRISPR-Cas13a System to Inhibit RNA Viruses

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.