Preliminary Research On The Construction Of Rabbit SMA Disease Model Based On Cas9 System

Spinal muscular atrophy(Spinal muscular atrophy,SMA)is an autosomal recessive genetic disease,which is caused by the deletion or mutation of the survival of motor neuron gene(Survival of Motor Neuron,SMN1).Proximal muscle weakness and muscle atrophy caused by metadegeneration.In this study,the rabbit SMN gene was knocked out based on the CRISPR/Cas9 gene editing system,and a rabbit SMA disease model was obtained,which provides a reference for the research and treatment of SMA disease.The main research results are as follows1.Analysis and cloning of rabbit SMN gene coding regionCompare the c DNA homology of SMN genes between rabbits and other mammals by bioinformatics analysis methods.The results show that the nucleotide sequence of the CDS region of the SMN gene is highly conservative.The homology between rabbits and humans,mice,monkeys and other species exceeds 80%.The total RNA of rabbit muscle tissue was extracted,and the c DNA sequence of rabbit SMN gene was amplified by RT-PCR.The sequencing results showed that the target gene obtained was consistent with the sequence of LOC100341643(survival motor neuron protein,SMN)gene on NCBI.The c DNA is 888 bp long,including 9 exons and 8 coding exons,encoding 295 amino acids.2.Analysis of the cleavage efficiency of the CRISPR/Cas9 system at the cellular levelSelect the four exons 1,3,4,and 7 of the SMN gene to design small guide RNA(sg RNA)in http://crispor.tefor.net/.Select 11 high-scoring sg RNAs to construct p X459-sg RNA recombinant vector and double fluorescent reporter RGS-sg RNA recombinant vector.Co-transfect the two recombinant vectors into293 T cells at a ratio of 1:1,and observe the transfection effect after 24 hours.And analyze the ratio of 293 T cells expressing green fluorescence and red fluorescence by flow cytometry,which is the gene editing efficiency of the target site.According to flow cytometry analysis,the top four sg RNA cleavage efficiency from high to low are sg RNA4-2(88.9%),sg RNA4-1(66.1%),sg RNA1-2(57.1%),and sg RNA7-2(56.8%).3.Preparation of CRISPR/Cas9-mediated SMN gene knockout rabbitsThe fertilized eggs were collected,and the in vitro transcribed sg RNA and Cas9 protein were microinjected into the rabbit fertilized eggs according to the1:5 premix.Cytoplasmic injection is divided into two groups: single sg RNA injection and double sg RNA injection.Among them,a single sg RNA was injected,and the sg RNA 4-2(88.9%)injection with the highest flow cytometry efficiency was selected.A total of 44 fertilized eggs were transplanted into 5 female rabbits.The pregnancy rate was 40%(2/5),the birth rate was 11.3%(5/44),and the survival rate of young rabbits was 100%(5/5).The gene editing efficiency of sg RNA was studied by the method of injecting two sg RNAs and four combinations.Cytoplasmic injection and autologous transplantation of a total of200 fertilized eggs into 30 female rabbits to obtain 34 young rabbits in the F0 generation.It was identified that in the combination of injected sg RNA4-2 and sg RNA7-2,the number Y0-3 is the SMN gene knockout rabbit.The results showed that the injection of single sg RNA and double sg RNA into female rabbits has no direct correlation with the pregnancy rate of female rabbits and the birth rate of young rabbits.Compared with the injection of double sg RNA,a single sg RNA injection can obtain a higher survival rate(100%)of young rabbits,while microinjection of double sg RNA can obtain SMN gene knockout rabbits more efficiently.In the 4 combinations of two sg RNA injections,the pregnancy rate of the female rabbits injected with the combination of sg RNA4-1 and sg RNA4-2 and the combination of sg RNA1-2 and sg RNA4-2 were both 50%,which was higher than the other two groups.Among the four combinations,the combination with the highest birth rate of newborn rabbits is the combination of injection of sg RNA4-1 and sg RNA4-2,and the survival rate of newborn rabbits is also.The birth rate of pups injected with double sg RNA is positively correlated with survival rate.In this study,a rabbit SMN gene was cloned by systematically analyzing the sequence of the SMN gene.Eleven sg RNAs were designed in exons 1,3,4,and7 of the gene,and the gene editing efficiency of sg RNA was systematically studied and optimized.Finally,the SMN gene knockout rabbit was successfully obtained by using the CRISPR/Cas9 gene editing system,which provided a basis for further research on the mechanism and treatment of SMA,and provided a reference for the construction of animal disease models through the CRISPR/Cas9 gene editing system.