Use CRISPR/Cas9 To Construct FAH Gene Defect Model Pigs

With the continuous development of science and technology,human beings need to understand various diseases thoroughly,and find effective ways to treat diseases.However,due to the limitation of ethics and morality,scientific research cannot directly use living people as the object of experimental research.Animals can be used as the direct object of scientific research,and scientists can conduct experiment directly on living animals that meet ethical review.Compared with rodents such as mice and rats,pigs are one of the species that are closer to humans in organ structure,size and physiological structure.Because of those advantages,they are widely used in scientific research.As an animal model,pigs have the most advantage.Since the beginning of gene editing technology,scientists have been able to use gene editing technology to obtain genetically edited animal,such as zinc finger nucleases?ZFN?technology,transcription activator-like effector nucleases?TALEN?technology and clustered regular interspaced short palidromic repeats?CRISPR/Cas?technique.Then,they conducts research on related diseases.At present,researchers have gradually established models of FAH disease targeting mice,rats,rabbits,and pigs through gene editing technology.Animals of these disease models have been identified to mimic human HT1 liver fibrosis,cirrhosis and other phenotypes very well.Hereditary tyrosinemia type I?HT1?is a serious autosomal recessive disorder in humans,caused by the deletion of the Fumarylacetoacetate hydrolase?FAH?in the patient.This enzyme is the last step to catalyze the metabolism of tyrosine.If the FAH gene is mutated,it will cause a large accumulation of ethyl fumarate and succinylacetone in the blood,which in the long run will cause serious damage to the liver and kidneys,and it can also cause liver and kidney disease in the fetal or early childhood,which eventually lead to death.In 1998,scientists found 2-?2-nitro-4-trifluoromethylbenzoyl?-1,3-cyclohexanedione?NTBC?can inhibit the accumulation of tyrosine catabolic products produced by ethyl fumarate and succinylacetone,and preventing liver and kidney toxicity.In 1993,Grompe M obtained the FAH mutant mice from gene-editing ES;in 2011,Raymond et al first obtained FAH^+/-model pigs by AAV vectors;in 2014,the same group obtained Female and male pig through somatic cell nuclear transfer?SCNT?,and the model pigs were mate sfter sexual maturity to breed FAH^-/-pigs;in 2016,Lijian Hui used the CRISPR/Cas9 technology to make FAH^-/-rats,and the symptoms were alleviated following intrasplenic injection with 10⁷ WT hepatocytes,;in 2017,Liangxue Lai obtained FAH^-/-model rabbits using TALEN technology.In this study,we constructed a FAH^-/-porcine fibroblast cell line using CRISPR/Cas9technology,and then successfully produced 10 FAH^-/-diseases model pig after embryo transfer by somatic cell nuclear transfer technology.The significance of this study is that we obtained the FAH^-/-model pig can directly by nuclear transfer technology without mating and breeding,and the reproductive cycle is shortened.Secondly,construction of FAH gene knockout porcine fibroblasts can be used to prepare FAH^-/-model pigs in large quantities by somatic cell nuclear transfer technology.The FAH^-/-model pig can not only be used as a disease model to study the pathogenesis and treatment of hereditary tyrosinemia,but also be the basic strain of the humanized pig model.In addition,It can also provide relevant models for clinical research to test the efficacy of various cell treatment methods and provide a glimmer of hope for solving the problem of organ shortage.?1?The FAH^-/-pig fetal fibroblast cell line was constructed using CRISPR/Cas9 technology.Constructing 6 fetal fibroblast cell lines with 35 days of fetus.After genotype identification,PEF-1,PEF-2 and PEF-6 are female,PEF-3,PEF-4 and PEF-5 are male.We designed a knockout vector for the sgRNA sequence in the first exon of the FAH gene,and established a FAH^-/-cell line by nuclear transduction.Then,we finally obtained a 40 bp knockout cell line FAH-31.?2?After somatic cell nuclear transfer and embryo transfer,we were obtain 10 FAH^-/-fetuses.Using FAH-31 as donor cell,10 FAH^-/-fetuses were obtained after somatic cell nuclear transfer and embryo transfer.Genotype identification confirmed that all births were consistent with the genotype of the FAH-31 cell line.?3?Using Western Blot technology,it was confirmed that the FAH protein was completely knocked out.?4?Immunohistochemistry and H&E staining techniques were used to detect liver and kidney phenotypes.The results showed that the liver tissue of FAH^-/-pig showed diffuse liver injury,necrosis of hepatocytes,enlargement of cytoplasm,large nuclear and significant nucleoli.The kidney of FAH^-/-pig has focal acute pyelitis,tubular dilatation and renal tubular epithelial damage,and the wild-type kidney structure is normal.?5?Through the routine blood test of peripheral blood,the contents of ALP,ALT,AST,TBA in FAH^-/-pig blood are significantly higher than those in wild-type pigs,which proves the Abnormal liver function and kidney function in FAH^-/-piglets.