Improved GC Efficiency And Precision Of Cytidine Base Editors And Their Application In Gene-edited Rabbit Models

According to the report,58% of pathogenic variations in human genetic diseases are attributable to point mutations in the genome.Gene edited animal models of specific disease loci are the basis of studying pathogenesis and developing therapies.However,there are a few flaws in the widely used CRISPR/Cas9-mediated homologous recombination technique: 1)The efficiency of point mutations is low(10%-20%);2)High proportion of Insertions and deletions(Indels)and other by-products;3)Deletion of large fragments in the genome.Cytidine base editor(CBE)is a novel genomic sitedirected mutation technique developed based on CRISPR/Cas9 system,which can achieve C-to-T base conversion at the target site.Compared to traditional CRISPR/Cas9-mediated homologous recombination,CBE produces C-T base conversion more efficiently and does not require additional DNA donor templates.More importantly,CBE does not induce double-strand DNA break(DSB),which greatly reduces the generation of Indels and other by-products and significantly improves the purity of products.Rabbit is a classical model animal.In our previous work,we used the CBE system to achieve efficient base editing in rabbit genome,and successfully constructed the albinism(Tyr nonsense mutation)and HGPS(Lmna RNA mis-splicing mutation)model rabbits.However,during the process,we found that the current CBE system still has some problems:1)Traditional r A1-BE based on rat APOBEC1 deaminase(r A1)has low efficiency in GC context,and it is difficult to obtain the mutant model of Tyr p.R299 H in in GC context;2)The main editing window of r A1-BE is large,consisting of ?5 nt,resulting in a large number of non-targeted C editing(Bystander mutations)at Tyr p.Q68 stop and Lmna p.G607 G in rabbits;3)When there are multiple consecutive Cs(CC context),the editing precision of r A1-BE is poor,and it is difficult to edit only a single C;Based on the above scientific problems,this study aimed to optimize the traditional r A1-BE to improve the GC efficiency and precision,and to construct the rabbit model of the corresponding disease loci by the optimized new CBE systems.First,we used AID deaminase to replace r A1,which prefer TC but disfavor GC sequence,and introduced hyperactive mutations and optimized codons to obtain e AIDBE4 max system.Compared with r A1-BE3 in rabbit embryos,e AID-BE4 max significantly improved the editing efficiency in GC context(30.58% vs.78.31%),and was no less efficient than r A1-BE3 in non-GC context.Tyr p.R299 H albino rabbits were successfully generated by e AID-BE4 max,and the average frequency of targeted mutation was 92%,much higher than that of traditional r A1-BE3 mediated rabbits(10%).Second,aiming at the low precision of r A1-BE3 due to its large window,we constructed a new YFE-BE4 max system by introducing appropriate point mutations into r A1,which can significantly narrow the window to 1-3 nt while maintain high efficiency.In addition,Tyr p.Q68 stop and Lmna p.G607 G rabbits significantly reduced bystander editing and improved editing accuracy.The rabbit models of the two loci also accurately simulated the typical pathological features of human albinism and HGPS,respectively.Finally,we designed the e A3G-BE4 max system using human APOBEC3G(h A3G)deaminase,which has a natural preference for CC context(underlined C).The e A3GBE4 max significantly prefers CCC>CCC>CC sequence,improves editing accuracy at continuous multiple C sites.In addition,e A3G-BE4 max achieves 18.3%-58.0% and54.5%-92.2% editing efficiency in human cells and rabbit embryos,respectively.We successfully obtained precisely edited Tyr p.Q48 stop albino rabbits by e A3G-BE4 max.In summary,this study developed new base editing tools to improve GC efficiency and precision by optimizing cytosine deaminases.The optimized CBE systems were used to efficiently and accurately mimic point mutations in rabbit models,providing ideal and new animal models for studying the pathogenesis of genetic diseases.