Evaluation Of Mutation Ability Of HBB Point Mutant Vector In Cynomolgus Embryo

?-thalassemia is a monogenic inherited hemolytic disease characterized by reduced,abnormal or absent synthesis of ?-globin chains.It has been reported so far that more than200 HBB gene mutations can cause ?-thalassemia.Because of its high prevalence and mutation complexity,it has been a hot topic of research.Cytosine base editors allow the conversion of cytosine-guanine(C-G)base pairs to thymine-adnine(A-T)by cytosine deaminase without double-strand DNA breaks.Compared to the base substitution induced by CRISPR/Cas system-mediated homology directed repair,the base editor increases editing efficiency and reduces indels,translocations and rearrangements caused by DNA double-strand breaks.In theory,correcting HBB mutations in human embryos through the base editor can prevent the disease transformation from parents to offspring.It brings about the possibility of curing genetic disorders such as ?-thalassemia.To test this hypothesis,an experimental animal model that mimics the human beta-thalassemia mutant genotype is needed.Therefore,in this study,we selected 7 HBB point mutation sites responsible for?-thalassemia.Take cynomolgus monkey embryos as the research object and use Sa BE4-Gam,Anc BE4 max,BE3 as tools to carry out gene editing.Based on the same mutation site,the editing effect of BE3 and An BE4 max was compared to evaluate the validity and feasibility of the Anc BE4 max on cynomolgus embryos.The main results are as follows:(1)Selection of point mutation sites in HBB gene and design of sg RNAsBased on PAM sequence requirements and mutational activity window constraints,7suitable mutation sites were selected from the relevant HBB point mutations listed in Clin Var.Coding region mutations HBB(364G>A)and HBB(47G>A);splice site mutationHBB(315+1G>A)and 5'UTR mutation HBB(-71C>T)and HBB(-50G>A),for the above 5PAM sequences are "NGG" mutation sites,the designed sg RNAs were named SP4,SP8,SP11 and SP42,SP43.Codon mutation point HBB(118C>T)and intron mutation HBB(93-21G>A),the sg RNAs designed for the above 2 PAM sequences with "NNGRRT" mutation sites were named SA7 and SA15.(2)Analysis of single base mutations in early embryos of cynomolgus monkeysAll sg RNAs were transcribed into m RNA in vitro,and the Anc BE4 max m RNA and the 5 sg RNAs with the PAM sequence "NGG" were respectively injected into the cytoplasm of the pronuclear fertilized egg,and 4 of the 5 sg RNAs showed editing activity.SP4 mutated at the target mutation site C6 with a mutation rate of 31%;SP11 showed mutation at C6 with a mutation efficiency of 40%;SP42 mutated at multiple editable C sites in the active window.The mutation efficiency of the target mutation site C7 was the highest(85%).The mutation of SP43 was found at C4,C5 and C12 sites.The mutation rate of C4 and C5 was 80%,and the C12 mutation rate was 25%.No obvious indel production and off-target were found.Sa BE4-Gam m RNA was respectively injected with 2 sg RNAs with a PAM site of "NNGRRT",and none of the 2 sg RNAs showed visible editing activity.(3)Comparison of BE3 and Anc BE4max4 sg RNAs m RNA that have been shown to be active by the Anc BE4 max were injected with BE3 m RNA.Mediated by the BE3,SP4 and SP43 did not show base editing activity.SP42 mutated at C7 site with mutation efficiency of 50% and homozygosity rate of 30%.SP11 had a mutation rate of 25% at C6 and homozygosity rate of 20%;mutation efficiencies and homozygous rates of both active sg RNA are lower than the Anc BE4 max.In conclusion,we successfully obtained 4 active sg RNAs mediated by Anc BE4 max,in which SP4 and SP42 can mutated at the ?-thalassemia mutation site,which can mimic the HBB genotype of human ?-thalassemia patients and lay a foundation for the establishment of a ?-thalassemia cynomolgus monkey model;SP11 and SP43 do not produce mutations at the target pathogenic mutation site,but the actual mutation production site may also lead to decrease or loss of ?-globin expression,which requires further experimental verification.Compared with the BE3,Anc BE4 max showed higher editing activity and mutation homozygosity,which provided an experimental basis for the application of the Anc BE4 maxeditor in embryos.