CRISPR/Cas9 Mediated Gene Knockout Of Chicken Muscle Growth And Melanogenesis Related Genes

CRISPR/Cas9 system is the third generation genome editing tool,which is composed of Cas9 protein and guide RNA recognizing the target DNA sequence.Taken advantages of specificity,efficiency and the simplicity of design,CRISPR/Cas9 was widely used for targeted genomic DNA editing in various species.Myostatin(MSTN)was proved to be a negative regulatory factor to the growth of skeletal muscle,and loss of this gene led to double-muscled phenotype,characterized by a large increase in muscle mass.Melanocortin 1 receptor(MC1R)and tymsinase(TYR)are key genes in melanogenesis in animals.In this study,four target sites(MT1,MT2,MC1 and TY1)were selected in the exons of MSTN,MC1 R and TYR based on the design principles of CRISPR/Cas9.Expression vectors and reporter vectors were constructed,and activities of four sets of CRISPR/Cas9 vectors were validated in HEK293 T cells.Chicken genomes were targeted edited by the CRISPR/Cas9 system in chicken DF-1 cells and fibroblasts isolated from chicken embryos.Optimization and exploration of injecting DNA into chicken blastoderm were carried out to establish injection system.The results achieved in the present study are displayed as follows.1.Four target sites(MT1,MT2,MC1 and TY1)were selected in the exons of MSTN,MC1 R and TYR by CRISPR design,and their expression vectors and reporter vectors were constructed by overlap PCR and direct annealing of two oligonucleotides,respectively.The activity of CRISPR/Cas9 nucleases was verified via co-transfection of expression vector and its reporter vector in HEK293 T cells.2.The cleavage efficiencies of CRISPR/Cas9 nucleases of MT1,MT2,MC1 and TY1 in chicken DF-1 cells after co-transfection with CRISPR/Cas9 vectors were 40.2%,37.6%,12.7% and 15.1%,respectively.The fibroblast cells were obtained by collagenase digestion from 10 to 11 days Lueyang black-bone chicken embryos.However,editing efficiencies of CRISPR/Cas9 system in fibroblast cells were too low to be detectable.3.Direct injection was utilized to deliver vectors into chicken embryos.We found that 2?L was the optimized volume.However,egg hatching rate successful significantly reduced after injection,and we speculated that the process of injection could damage chicken embryos.The integration efficiency of lentivirus LV-Cas9-Puro into chicken genome was about 8.70%.No gene editing events of TYR were found after injection of CRISPR/Cas9 expression vectors.In summary,we successfully constructed four specific target vectors of CRISPR/Cas9 system and reporter vectors for MT1,MT2,MC1 and TY1 sites.Target gene modifications were achieved in chicken DF-1 cells and fibroblasts isolated from Lueyang black-bone chicken embryos.Additionally,the optional injection volume was confirmed for chicken embryo direct injection.And the lentivirus was successfully inserted into the chicken genome,however,target modifications via CRISPR/Cas9 system in chicken embryos were not found.Collectively,this study provided basic data and technical support for improving chicken gowth traits,enhancing melanogenesis and breeding.