Knockout Of Chicken TVB Gene By Multiplexed CRISPR Technologies And Establishment Of A New Multiple Gene Editing

Avian leukemia(avian leukosis)is considered to be one of the essential causes of severe economic losses in the poultry industry,but there are no effective means to prevent and cure the disease.Chicken TVB(tumor virus locus B)is a receptor gene that controls the invasion of three subtypes of avian leukemia virus(avian leukosis virus)B into the host.In this study,the multiple gene-editing techniques of regularly spaced short palindromes(clustered regularly interspaced short palindromic repeats,CRISPR)was used to study the gene knockout of chicken TVB locus,the efficiency of this technique was compared with that of conventional CRISPR gene-editing technique,and the feasibility of preparing TVB gene knockout chicken was further explored.In addition,this study preliminarily established a novel CRISPR multi-gene editing system based on the overexpression of CRISPR-associated protein 9(Cas9)and the in vitro transcription of multi-guide RNA,which improved the convenience of multiple gene editing.The results of the study are as follows:1.Knockout Chicken TVB Gene by CRISPR multiple Gene Editing techniqueEight guide RNA target sequences of the Cas9 gene-editing system were designed for the second and third exons of the TVB gene.The cleavage activity of the above eight guide RNA was proved by the SSA reporting system in HEK293 T cells.Plasmids expressing single guide RNA and multiple gene editing plasmids simultaneously expressing four guide RNA were constructed and transfected into chicken DF-1 cell line puromycin drug sieve.T7E1 digestion showed that multiple gene-editing mediated by four guide RNA could significantly improve the knockout efficiency of TVB gene.Plasmids expressing single guide RNA and multiple gene-editing plasmids simultaneously expressing four guide RNA were constructed and transfected into the chicken DF-1 cell line puromycin drug sieve.T7E1 digestion showed that multiple gene-editing mediated by four guide RNA could significantly improve the knockout efficiency of the TVB gene.The adenovirus vector-mediated Cas9 multiple gene-editing was injected into a chicken embryos by microinjection,and it was found that it could achieve efficient gene knockout in living chicken embryos,which proved that CRISPR multiple gene-editing had the potential to prepare gene editing chickens.2.Establishment of a novel CRISPR/Cas9 multiple Gene Editing systemTo overcome the tedious editing process of Cas9 multiple gene based on plasmid or virus expressing multiple wizard RNA at the same time,this study used Piggy Bac transposon plasmid expressing Cas9 protein and reporter gene EGFP,combined with puromycin drug screening,to prepare HEK293 T cell line with overexpression of Cas9/EGFP,and determined that the copy number of exogenous inserted gene was 6.There was no significant difference in cell proliferation activity between Cas9/EGFP and control cells.Three and four target sequences were designed for the endogenous gene FANCF and multi-copy foreign gene EGFP,respectively,and the guide RNA was prepared by in vitro transcription.The cleavage results in vitro showed that the above guide RNA had targeted activity.The RNA transcribed in vitro was transfected into the Cas9 overexpressed cell lines by liposome.After T7E1 digestion and fluorescence intensity detection,it was found that this method could achieve efficient multiple gene editing for both single-copy gene FANCF and multi-copy gene EGFP.The above studies confirmed the high efficiency of CRISPR multiple gene editing in knockout chicken TVB gene,they laid a foundation for the subsequent preparation of avian leukemia resistant chickens with TVB gene knockout.The new CRISPR multiple gene-editing system based on Cas9/EGFP overexpression monoclonal cell line and RNA in vitro transcription guides large-scale gene-editing research in specific cells.