Engineering CRISPR immune systems conferring GLRaV-3 resistance in grapevine

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the causal agents of grapevine leafroll disease (GLD), which severely impacts grapevine production in most viticultural regions of the world. The development of virus-resistant plants is a desirable strategy for the efficient control of viral diseases. However, natural resistant resources have not been reported in the genus Vitis, and anti-GLRaV-3 research has been quite limited in grapevine. In this study, by expressing FnCas9 and LshCas13a, we established a highly effective transgenic construct screening system via an optimized Agrobacterium-mediated transient delivery system in grapevine plantlets. Our study indicated that CRISPR/FnCas9 and LshCas13a caused GLRaV-3 inhibition. Moreover, three vectors—pCR01-CP, pCR11-Hsp70h and pCR11-CP—exhibited the most robust inhibition efficiency compared to those targeting other sites and could be further engineered to generate GLRaV-3-resistant grapevine. In addition, the viral interference efficiency of FnCas9 was dependent on its RNA binding activity. The efficiency of virus inhibition was positively correlated with the level of Cas gene expression. Importantly, we demonstrated that LshCas13a had better interference efficiency against viruses than FnCas9. In summary, this study confirmed that these two RNA-targeting CRISPR mechanisms can confer immunity against viruses in grapevine, providing new avenues to control GLRaV-3 or other RNA viruses in fruit crops.