CRISPR/Cas9-mediated Chicken Growth Gene Editing In Vitro

The growth of bones and muscles is the most important concern in the production of broilers.The broiler producer has been trying to increase muscle growth and meat production capacity.Myostatin can negatively regulate muscle growth,so it can be increased by knocking out the gene.Myocyte enhancer factor 2 plays a key role in skeletal muscle development.In this experiment,the interval repeats of the rDNA gene were used as target sites to allow the MEF2 gene to be located in an active transcription unit and increase insertion efficiency.The MSTN gene was knocked out at the same time.This will provide a reference for further exploration of quality transgenic chickens.Using bioinformatics analysis,the subject identified a site suitable for g RNA recognition and combining Cas9 cleavage in the internal transcribed spacer sequence between chicken r RNA genes,and designed a sequence for g RNA based on the sequence characteristics of g RNA.g RNA sequences and construction of eukaryotic expression vectors that simultaneously express g RNA and Cas9.Two gene-targeting cognate sequences DS1 and DS2 were designed to be amplified on both sides of the g RNA-recognized target site,and DS1 and DS2 were inserted into the synthesized pEGFP-MEF2 C vector to construct pEGFP-DS1-MEF2C-DS2 gene targeting vector was used for the insertion of the MEF2 C gene.For the MSTN gene to be knocked out,g RNA was used to design the website design g RNA sequence,and then g RNA and Cas9 eukaryotic expression vectors were constructed.The target vectors of MSTN gene and MEF2 gene were transfected into chicken fibroblast cells,and the green fluorescent protein was expressed in the cells.After that,the gene was cut and inserted.In this study,the MSTN gene Cas9 knockout vector and MEF2 C insert gene vector were successfully constructed,and expanded culture and massive extractionwere performed.The Cas9 expression vector was transfected into chicken fibroblast cells respectively.After 72 hours,the recognition site sequence was amplified.Cas9 could be used to preliminarily determine that genomic DNA could be cut accurately.The base sequence mutation was found in the fragments near the Cas9 recognition sequence.Six of the sixteen sequencing samples were mutated and the cleavage efficiency was 37.5%.In the editing experiment of rDNA,9 of the 25 sequencing samples were mutated with an efficiency of 36%.In the detection of the effectiveness of the MEF2 C gene targeting vector for gene integration,gene integration was found.Detection found that MEF2 c gene site-specific integration.In summary,The MSTN gene was successfully knocked out in chicken fibroblasts and the MEF2 c gene was inserted at a fixed point.