Safety Evaluation Of MSTN Gene-edited Tan Sheep

MSTN is a negative regulator of muscle growth and development.Targeted editing of MSTN by genome editing technology can inactivate its protein expression,increase the number and diameter of muscle fiber,and then promote muscle growth of livestock and poultry.Because of its advantages of simple and efficient,the CRISPR/Cas9 technology has been widely applied in biological breeding,greatly accelerate the breeding process.Now,in the United States and Japan,the genome edited animals and products have gradually stepped into industrialization,but the public still doubt about the safety of these animals.It is helpful to realize the industrialization development of genome edited animals in China by comprehensive safety evaluation to eliminate the public’s concerns.In this study,based on the MSTN gene-edited Tan sheep prepared in the early stage and the Guidelines for Safety Evaluation of Transgenic Animals,we set up the safety assessment procedure of gene-edited Tan sheep and comprehensively and systematically evaluate the safety of MSTN gene-edited Tan sheep from the aspects of living body safety assessment,mutton safety assessment and environmental safety assessment.1.Living body safety assessment of MSTN gene-edited Tan SheepThe evaluation contents of this experiment included genotype identification of the MSTN gene-edited Tan sheep,MSTN transcriptional expression identification,prediction of amino acid sequence after MSTN edited,off-target detection,physiological and biochemical indicators detection and gut microbial metagenomic analysis.The results showed compared with wild-type Tan sheep,the MSTN of F0 gene-edited Tan sheep produced 3bp deletion in the second exon and 11 bp deletion in the third exon,and the mutation could be transfered to offsprings.Gene-edited Tan sheep could produce m RNA and the level of the transcriptional product did not change significantly.The edited MSTN had a frameshift mutation,which resulted in premature termination of protein translation.rectal temperature and heart rate of gene editing Tan sheep did not change significantly,but respiratory rate increased significantly(p=0.022);the creatinine(p=0.001),alanine aminotransferase(p=0.047)and cholinesterase(p=0.002)contents in blood of gene-edited Tan sheep were significantly increased;the overall composition of gut microflora of gene-edited sheep did not change significantly,but the abundance of Firmicutes increased significantly(p=0.039 in male sheep,p=0.023 in female sheep).2.Mutton safety assessment of MSTN gene-edited Tan SheepThe evaluation contents of this experiment included slaughter performance assessment,meat quality analysis,tissue and organ pathological section analysis and 90-day rat feeding experiment.The results showed compared with wild type Tan sheep,the living weight(p=0.016),carcass weight(p=0.005)and eye muscle area(p=0.003)of MSTN gene edited Tan sheep were significantly increased.The meat color,p H,water loss rate,cooked meat rate and intramuscular fat of mutton did not change significantly.The gluteal muscle,heart,liver,spleen,lung and kidney did not observe any pathological changes.Compared with the control groups,the clinical observation indexes of the experimental groups were normal,the growth and development did not change significantly,and there was no abnormal phenomenon of blood physiological and biochemical indexes and urine biochemical indexes caused by feeding the diet containing gene edited mutton.3.Environmental safety assessment of MSTN gene-edited Tan SheepThe evaluation contents of this experiment included the detection of horizontal gene transfer between Tan sheep population and between Tan sheep and soil microorganism.The results showed that the mutated MSTN was not transferred horizontally from gene-edited sheep to wild-type sheep,and the MSTN was not detected in the soil microorganisms around the sheep pens.The above results show that the living body safety and mutton safety of MSTN geneedited Tan sheep are no different from those of wild-type Tan sheep,and gene-edited Tan sheep have no adverse effects on the surrounding environment.