CRISPR/Cas9 System-based Gene Editing Of The LAT-Clustered Micro Rnas And Its Influence On In Vitro Replication Of Marek's Disease Virus

Marek's disease virus is cell-bound virus,which can infect the natural host,such as chicken,turkey and quail,and induce the proliferation of host lymphoid tissue,leading to the occurrence of Marek's disease.MDV spreads horizontally in chickens through air-source medium,causing immune suppression and increasing susceptibility of chickens to other pathogens.MD is highly contagious and usually kills large numbers of chickens,causing huge economic losses to the world's poultry industry.MD is the first viral oncologic disease that can be prevented by vaccination with attenuated vaccine.However,the virulence of MDV epidemic strain has been continuously enhanced because of the long-term intensive breeding mode and the long-term immune pressure,which has broken through the existing vaccine protection and led to the local outbreak of the disease.Research on the function of MDV gene,pathogenesis and tumorigenesis mechanism will play a vital role in the prevention and control of MD.Micro RNAs are a class of endogenous non-coding small RNAs,about 20~25 nt in length,which play an important role in gene regulation after gene transcription.Mi RNAs were found in different serotype of MDV.MDV-1 coding 14 precursor of mi RNAs,processing into 26 mature mi RNAs.According to mi RNAs' relative positions in the genome,mi RNAs was divided into three typical mi RNA gene clusters(Meq,Mid and LAT).The Meq and Mid gene clusters are located in the Internal repeat short region of MDV,and are located in the upstream and downstream of meq respectively,while the LAT-cluster is located in internal repeat short region.Previous studies have shown that mi RNAs in both Meq-cluster and Mid-cluster are non-essential for MDV-1 replication and immunosuppression,but the potential biological function of the LAT-cluster remains unclear.Since mi RNAs of LAT-cluster are located in the first intron of latency-associated transcript,it is speculated that LAT-cluster may be associated with latency-associated viral infection.In this study,we successfully generated a LAT-mi RNA-deleted mutant of GX0101,a very virulent MDV strain,laying a foundation for clarifying the relationship between LAT-cluster mi RNAs and MDV pathogenicity,tumorigenicity and latent infection.According to the reported sequence of vv MDV GX0101 genome,eight guide RNAs of LAT-cluster were designed and cloned into the p X459 vector to construct the px459-g RNA plasmid.LAT-cluster mi RNAs were edited by plasmid transfection and virus infection.The gene deletion strains were screened by PCR amplification and identified by sequencing.Biological identification was performed on the obtained gene editing strains of GX0101,including passage stability analysis,virus proliferation curve,IFA staining and q PCR analysis.In this study,we successfully generated a LAT-mi RNA-deleted mutant,namely GX(?)LAT-mi Rs-C21-15.The mutant virus displayed good stability in passages and mi RNA deletion did not affect in vitro virus replication or the normal expression of randomly selected viral protein-coding or mi RNA genes.In conclusion,this study successfully generated a LAT-mi RNA-deleted mutant by CRISPR/Cas9 technology,which provided experimental materials for further research on the molecular regulation mechanism of LAT-cluster mi RNAs,and also laid a good experimental foundation for animal challenge experiments to study the function of LAT-cluster mi RNAs.