Construction Of Mutant Chickens Via PiggyBac Transposon And CRISPR Library

The chicken has historically been an important animal model for the study of developmental biology.Gene modified chicken models are of great significance for several reasons.First,chicken oviduct can be modified as a promising bioreactor for protein production,especially those with enormous pharmaceutical value.Second,new genomic engineering technology will greatly benefit poultry breeding for production of chickens with better agricultural production traits.Third,gene-knockout chicken models will provide better opportunities to explore genetic mechanism underlying various developmental events.Yet,the use of chicken in research has long been hindered by lack of efficient genetic engineering methods to manipulate the chicken genome.Currently chicken genetic engineering is facing the following challenges:1)difficult in vitro culture of germline competent PGCs and lack of efficient gene engineering means;2)great technical difficulty and long preparation period of homozygous mutants;3)lack of stable male sterile line as the recipient;and 4)low germline transmition efficiency.The current study aims at developing a stable culture system of chicken PGCs via optimizating culutre conditions,construction of piggyBac transposon mediated CRISPR/Cas9 sgRNA library targeting the whole chicken genome and preparation of large-scale mutants,and creating an inducible cell ablation system in chicken combining Tet-On and TMP dual regulatory systems.In our study,a stable in vitro culture system of chicken PGCs was first established through optimizing the culture and isolation conditions such as culture medium choices,and treatment conditions of different feeder cells.We successfully obtained 39 PGCs cell lines in vitro derived from White Leghorn chicken embryo gonads,Nongda3 chicken embryonic germinal crescent region and gonadal tissue.These cell lines had self-renewal property in vitro,were alkaline phosphatase positive,and had a normal diploid karyotype.The results of RT-PCR showed that the germ cell specific CVH and DAZL genes were expressed in these PGCs.The immunofluorescence results of PGCs further showed that CVH and DAZL were positive,the surface antigen SSEA-1 and SSEA-4 of stem cells were highly expressed,and SSEA-3 was weakly expressed.In addition,the stable transfection of PGCs was achieved successfully by piggyBac transposon system,the G0 germline chimeras were obtained by microinjection,and the G1 positive individuals were obtained by breeding sexually mature G0 chimeras with wildtype hens.Southern Blot and Genome Walking technology were used to analyze the positive G1 individual,and the results demonstrated that the foreign gene was single copy insertion,and the insertion site was located in the fourth intron of Fam20C gene on chicken chromosome 14.We next designed and synthesized 93961 sgRNAs targeting 16821 chicken protein-coding genes(approximately 5-6 sgRNAs per gene).We used a piggyBac vector to express these sgRNAs in PGCs that were simultaneously transfected with an inducible Cas9 vector.After obtaining a large number of mutant PGCs,the PGCs were injected into the recipient embryos,and the chimeras containing the mutant cells were generated.We found that the expression of Cas9 was regulated by DOX in PGCs and individuals.Moreover,102 different kinds of sgRNAs were detected by deep sequencing of testis DNA of G0 chimeras,and they were evenly distributed on the chicken chromosomes.Furthermore,we used Tet-On and TMP dul-regulation system to achieve specific cell ablation in PGCs.In summary,the G0 modified chimeras and G1 positive individuals were successfully generated by optimizing the culture conditions of PGCs.With the sgRNA library of chicken CRISPR/Cas9,the G0 chimeras containing different types of sgRNAs were obtained,laying the foundation for the large-scale preparation of mutant chickens in the future.Combining Tet-On and TMP regulatory systems,specific cell ablation of PGCs was achieved,providing a new approach for efficient germline transmission of modified PGCs using male sterile recipient chicken.