Knocking Out Chicken GDF8 Gene Using CRISPR/Cas9 Technology

Poultry is an important source of animal protein for human beings.Improving feed conversion efficiency and growth rate of poultry is a goal that poultry farmers and scientists continue to explore and pursue.Growth differentiation factor 8(GDF8,also known as MSTN)is highly expressed in animal muscle tissue and is a negative regulator of skeletal muscle growth and development.The mutation of GDF8 gene can cause the"double muscle"phenotype of animals.Therefore,the regulation of GDF8 gene by biotechnology could be a strategy to improve the meat production performance and growth rate of animals.CRISPR/Cas9 is currently the most commonly used gene editing method due to its advantages of high precision,efficiency and simplicity.In this study,we used CRISPR/Cas9 gene editing technology to explore the editing strategy suitable for poultry gene,and developed GDF8 gene-deficient transgenic germline chimeric chickens.First,in order to understand the evolutionary characteristics and functions of the chicken GDF8 gene,we performed homology analysis on the GDF8 gene sequence of poultry and mammals.The analysis results show that the GDF8gene has evolved conservatively among various species,and it is speculated that the GDF8 gene of poultry and mammal performs the same function.The functional enrichment analysis of chicken GDF8 gene also showed that the function of chicken GDF8 gene is the same as mammals,which is mainly the repair of post-traumatic injury.The expression of GDF8 gene in different tissues of chicken embryo was identified by quantitative PCR.It was found that GDF8gene was highly expressed in muscle tissues such as legs and chest,which confirmed that chicken GDF8 gene is closely related to skeletal muscle growth and development.Second,to achieve efficient editing of the GDF8 gene in poultry cells,we optimized the CRISPR/Cas9 system.In this study,four sg RNAs were designed for exon 1 of GDF8 gene,which were constructed into px459 plasmid,and transfected into DF-1 for gene knockout.It was found by T7E1 ligase detection that all the four constructed PX459-sg RNA plasmids could achieve GDF8 gene knockout,among which PX459-sg RNA2 knockout efficiency is the highest.In order to explore the higher knockout effect,this study combined 3 sg RNAs into the same plasmid-PX459-multisg RNA.After verification,the PX459-multisg RNA plasmid achieved better knockout effects than single sg RNA on both DF-1 and PGCs.By picking a single positive cell for PCR sequencing,which showed that PX459-multisg RNA could achieve gene knockout effect on chicken GDF8 gene from several bases to large fragment deletion.Third,in order to track the preparation of GDF8 mutant chicken,this study used the method of site-specific insertion of m Cherry fluorescent reporter gene to achieve GDF8 gene knockout.First of all,we compared three gene integration strategies:HMEJ(Homology mediated End Joining),HR(Homology directed Repair)and HITI(Homology-independent targeted integration)to optimize the CRISPR/cas9-mediated site-specific integration method of gene.By co-transfecting GDF8-HMEJ,GDF8-HDR and GDF8-HITI donor plasmids with Cas9-sg RNA2 into DF-1 cells respectively,after 15 days of transfection,PCR results for three groups of positive cells showed three strategies can achieve site-specific integration of genes.At the same time,the proportion of m Cherry~+cells detected by flow cytometry in the three groups was13.75%in the HMEJ group,2.14%in the HDR group and 1.23%in the HITI group.This shows that the HMEJ strategy is more efficient than the integration of HR and HITI.Finally,we used the PGC cell-mediated method to prepare GDF8 knockout chickens.The GDF8-HMEJ and Cas9-sg RNA2 plasmids were co transfected into the primary PGCs of chicken,and then m Cherry+PGCs were obtained by flow cytometry separation twice.GDF8 mutant m Cherry+PGCs were injected into recipient chicken embryos that developed to H15 stage by chicken embryo injection.A large number of m Cherry+cells were observed in the right and left gonads of recipient chicks after 7 days of injection,which indicated that the germline chimeric chicken embryo with GDF8 gene defect was successfully prepared in this study.By further incubating chimeric chicken embryos,we eventually obtained seven adult germline chimeric chickens with GDF8 gene defects.In summary,this study proves that multiple sg RNA-mediated CRISPR/Cas9 technology can cause the knockout effect of large deletions of chicken GDF8 gene.At the same time,the donor plasmid of GDF8 gene exon 1 was constructed by HMEJ integration strategy,and it was initially applied to the preparation of transgenic chicken mediated by PGC method to obtain GDF8mutant germline chimeric chicken.The research results laid a foundation for studying the role of GDF8 gene in improving meat quality and growth performance and breeding new varieties.