T158M Single Base Editing Of MECP2 Gene In Murine And Rhesus Monkey’s Embryos

Rett syndrome(RTT)refers to the disease that seriously affects the psychomotor development of children,especially female(male embryos are mostly lethal).MECP2 gene mutation is the direct cause of RTT(nearly 70% of RTT patients have MECP2 gene mutation,and MECP2 mutation has a 90% chance of causing RTT).But due to the lack of ideal animal models,there is currently no effective treatment for RTT.The mutation mode of this gene is caused by the change of single nucleotide C→T with80% probability.Hundreds of MECP2 mutations causing RTT have been identified,of which 8 hotspot of mutations account for more than over 60% and T158 M point mutation dominates.As mice produced by Cre-loxp technology lack MECP2 and cannot be used to simulate patient’s behavioral movements and there is dissimilarity in human brain structure and genetic background between rodents and humans,non-human primates(such as macaques and cynomolgus monkeys)is particularly important to build animal models of RTT diseases.In 2014,our laboratory first reported the successful use of the targeted gene editing method TALEN to edit the MECP2 gene monkey model.Existing studies found that the primate model is more similar to the clinical disease phenotype,which can effectively simulate social disorders,stereotyped behavior,abnormal brain development and other phenotypic data that are difficult to obtain in rodent models.However,in the models obtained based on TALEN technology,many types of mutations exist,and mutation rate is low.Therefore,obtaining a single point mutation type primate animal model is expected to avoid these problems.In this study,with the use of BE system and theclinical hot spot mutation T158 M as the target,the optimal editing scheme was verified and screened at the cell and mouse embryo level,and tested at the primate embryo level,and an effective editing scheme was obtained.It lays the foundation for the establishment of RTT single hotspot mutant monkey model.The research results are as follows:1.sgRNA design and Screening of at the cellular levelFirst,the T158 M of exon 3 of the MECP2 gene was selected as the target,and three pairs of sg RNA oligonucleotide chains for this site was designed by using software,which were connected to the PGL3-U6-puro plasmid respectively.Thus the constructed sg RNA plasmid was successfully obtained by sequencing analysis.Commercialized 293 T cells were chosen,and the sg RNA plasmid and BE plasmid were co-transfected into 293 T cells by lipofection.The effective sg RNA was screened by observing the fluorescence rate and sanger sequencing of the cell genome.The C→T mutation at the T158 M site of exon 3 of the MECP2 gene was successfully achieved,and the effective sg RNA sequence was Target-1.2.In vitro transcription quality control and optimization of mouse embryo concentration combinationUse T7 in vitro transcription kit to transcribe sg RNA,BE3 and BE4 max,and then recover the m RNA obtained by sg RNA、BE3 and BE4 max.Dilute it to the required concentration for microinjection,and grow it into early embryos in vitro.The BE editing efficiency and its effect on embryo blastocyst development rate could be observed.Firstly,the macaque embryo was used to detect the quality of the in vitro transcribed m RNA,and the problem was successfully solved.After quality control of in vitro transcription,four concentration combinations were designed using mouseembryos(BE4max-P2A-GFP: T158M-sg RNA = 20:10,50:20,100: 50,and 200:100),and 3 For the second iteration,when embryos were injected with a combination of BE4max-P2A-GFP: T158M-sg RNA = 100: 50,the mouse embryo blastocyst rate and editing efficiency were the best.3.Rhesus monkey embryo development and editing statisticsUsing the micromanipulation technique,the 100: 50 concentration combination was injected into the cynomolgus monkey embryo,and the gene editing embryo blastocyst rate and efficiency were analyzed.The mutation rate of embryos found:homozygous mutation accounted for 12.80%,chimera mutation accounted for 18.00%,wild type accounted for 69.20%.The above results show that C→T transition at position 158 of the MECP2 gene in mouse and macaques embryos were achieved,establishing a stable embryo editing system for the later establishment of a T158 M mutant RTT animal model.