The Construction Of PVY RNA Edited System Based On CRISPR-Cas13d

Potato virus Y（PVY）is one of the most destructive viral pathogens,affecting crop productivity on a worldwide scale.It has a significant impact on the production and quality of agricultural crops.It occurs often in agricultural planting regions across the globe,and the extent and degree of harm is growing.There are no effective preventions and control methods.The CRISPR-Cas system is the first line of defense against viruses for microorganisms.It consists of Cas protein with binding sites and sg RNA.The Cas13d protein can cleave the specific sequence with the guidance of the specific sg RNA.The CRISPR-Cas13 system offers a novel strategy for the prevention and control of RNA viruses.To determine whether the CRISPR-Cas13d system can accurately cut the RNA virus strand and if it may be utilized as a novel method to prevent RNA viruses like PVY.In this work,a TRV-CRISPR-Cas13d vector system was created based on the CRISPR-Cas13d system to stably express the Cas13d-sg RNA protein complex in plants,and the RNA editing effectiveness of this system was compared and assessed.A novel preventive and control method for the PVY virus was also presented.The main findings are following:（1）Two recombinant vector systems for delivering CRISPR-Cas13d have been successfully created with molecular biology,gene recombination,and other techniques--TRV-CRISPR-Cas13d for stably expression and p Earley Gate100-CRISPR-Cas13d for temporary expression.It was determined that both TRV-CRISPR-Cas13d and p Earley Gate100-CRISPR systems can effectively express Cas13d protein in plants with RT-q PCR and Western-Blot.It was determined that both p Earley Gate100-CRISPR-Cas13d and TRV-CRISPR-Cas13d are capable of expressing Cas13d protein in plants by observing the expression of Cas13d fusion protein GFP using laser confocal microscopy.（2）Using GFP as a research object,the targeting efficiency of the TRV-CRISPR-Cas13d^GFP、p Earley Gate100-CRISPR-Cas13d^GFP、and VIGS^PDS systems to foreign genes were compared.It is evident that the TRV-CRISPR-Cas13d^GFP system has a greater knockout efficiency for GFP than the p Earley Gate100-CRISPR-Cas13d^GFP system,based on the detection of GFP signal in conjunction with RT-q PCR,Western-Blot and other technical methods.Notably,three distinct sg RNAs were designed for the GFP gene,yet the performance of a single sg RNA was superior.（3）Taking PDS as the research object,the interference efficiency of TRV-CRISPR-Cas13d^PDS and VIGS^PDS was compared.Based on plant phenotypic,it was found that both systems can lead to obvious leaf whitening phenomenon.It is proved that both the TRV-CRISPR-Cas13d^PDS system and the VIGS^PDS system can systematically and continuously target plant endogenous gene PDS and lead to the leaves of the treated plants were whitened,but there were differences in the leaf whitening phenotype.The degree of whitening of the VIGS^PDS system was better than that of the TRV-CRISPR-Cas13d^PDSsystem.Further analysis with RT-q PCR and transcriptome sequencing proved that TRV-CRISPR-Cas13d^PDS mediated higher PDS gene interference efficiency and lower off-target rate.（4）The PVY genome data registered in the NCBI database was systematic analyzed,representative PVY strains were selected,MUSCLE software was used for sequence analysis,the sequence of the conserved regions of PVY was determined,and several sg RNAs were screened out,further two specific target sequences for sg RNAs were identified.By observing the PVY-GFP signal and also combined with RT-q PCR and Western-blot,it was confirmed that the TRV-CRISPR-Cas13d^PVY system can significantly inhibit the replication and proliferation of PVY.The e IF4E1 is necessary for the infection of Potyvirus.In this study a sg RNA specific to N.benthamiana e IF4E1 was designed and constructed TRV-CRISPR-Cas13d^{PVY+e IF4E1},which can target e IF4E1 and PVY-CP at the same time.Compared with the TRV-CRISPR-Cas13d^PVY system,the TRV-CRISPR-Cas13d^{PVY+e IF4E1} system showed higher efficiency.TRV-CRISPR-Cas13d^{PVY+e IF4E1} also has significant resistance to Tu MV,which belongs to the Potyvirus.This system provided a research basis for the precise prevention and control of Potyvirus virus disease,and also provided reference for the prevention and control of other RNA viruses.