Application Of CRISPR/Cas13 System For Resistant Engineering Of Potato RNA Viruses And Regulation Of Chloroplast Genes In Tobacco

The prokaryote-derived CRISPR-Cas genome editing technology has the thepotential to revolutionize plant breeding,and improve crop production.Among the currently major CRISPR-Cas types,the type VI CRISPR-Cas13 is unique in its ability to exclusively bind and cleave foreign ss RNA.In higher plants,Cas13 has been used to combat pathogenic RNA viruses or regulate genes expression,knockdown m RNA and act as nucleic acid detection tool.Here,we mainly provide an overview of the diverse application of CRISPR/Cas13 system in different eukaryotic fields.Our research reports on the repurposing of CRISPR/Cas13 system for protectection of potato plants from major potato RNA viruses,and for regulating genes expression in tobacco chloroplast.The main results as following:1.Engineering high-level potato viruse Y(PVY)resistance into potato plants by introducing the CRISPR/Cas13 a prokaryotic immune system.Transgenic potato lines expressing Cas13a/g RNA(single guide RNA)constructs showed suppressed PVY accumulation and disease symptoms.The levels of viral resistance correlated with the expression levels of the Cas13a/g RNA construct in the plants.Further the data demonstrated that appropriately designed g RNAs can specifically interfere with multiple PVY strains,while having no effect on unrelated viruses.2.Repurposing of CRISPR/Cas13 a coupled with an endogenous t RNA-processing system(polycistronic t RNA-g RNA,PTG)to produce multiple g RNAs with varying expression levels that target four genes of PVY.Transgenic plants expressing multiple g RNAs displayed similar suppression of PVY accumulation and reduced disease symptoms as those expressing a single g RNA.Moreover,Cas13a/PTG-transformed plants with different expression levels of multiple g RNAs displayed similar resistance to PVY strains.Collectively,data revealed that the number and expression levels of g RNAs had no significant effect on CRISPR/Cas13a-mediated viral interference in plants.3.Developing a Cas13d/PTG system for engineering broad-spectrum resistance to RNA viruses in potato.The Cas13d/PTG system enabled programmable RNA virus interference for targeting either one virus alone or two mixed RNA viruses simultaneously.This extended the applicability of the CRISPR system to multiple RNA virus resistance for crop protection.4.Establishment of the CRISPR/Cas13 d system in tobacco chloroplasts and utilizing CRISPR/Cas13 d to regulate gene expression.Generation of desired g RNAs with ribozyme sequences flanking the designed g RNA by plastid transformation.However the editing efficiency for chloroplast endogenous genes remain further study.In sum,our research had identified some limits of CRISPR/Cas13 system for interference with potato virus Y,and also provided an efficient strategy for engineering broad-spectrum resistance to multiple RNA virus infection in crops.Thereby this extended the applicability of the CRISPR system to multiple RNA virus resistance for crop protection.Meanwhile,the CRISPR/Cas13 system was established in tobacco chloroplasts and the editing efficiency for chloroplast endogenous genes was preliminary evaluated.Our data laid a foundation for the application of CRISPR/Cas13 in chloroplasts.