The Optimization And Application Of Base Editing Technology

Single base editing techniques,including cytosine base editors(CBEs)and adenine base editors(ABEs)were developed by the fusion of cytosine deaminase or adenine deaminase with Cas9.CBEs and ABEs enabled to convert cytosine(C)to thymine(T)or adenine(A)to guanine(G),respectively,within a 5-nt sequence editing window(typically,4-7)in genomic DNAs.Compared with the traditional cas9-mediated homologous recombination base substitution technique,base editors have the advantage in high base editing efficiency with no double-stranded DNA breakage.Currently,they have been widely used in gene editing,gene therapy,animal disease animal model,drug screening and crop breeding,etc.Base editors are powerful genome editing tools,However,some limitations for base editors still existed: For example,limited base editing widow and low base editing efficiency for Cs proximal to PAM for CBEs hindered its boarder applications;Current base editors can only convert either adenines or cytosines.In this study,a series of new base editors were developed through protein engineering,which improved base editing activity,expanded the base editing window and base editing product,and provided new ideas for the optimization and application of base editing tools.First,given the characteristic of single stranded DNA as the substrate of cytosine deaminase,through insertion of a non-sequence-specific single-strand DNA binding domain(ssDBD)from Rad51 protein between Cas9 nickase and the deaminases,serial hyper CBEs(hyCBEs)were generated with substantially increased activity and an expanded editing window toward the protospacer adjacent motif(PAM)in both cell lines and mouse embryos.Additionally,hyeA3A-BE4 max selectively catalyzed cytidine conversion in TC motifs with a broader editing range and much higher activity(up to 257-fold)compared with eA3A-BE4 max.Through injecting hyCBEs/CBEs mRNA and sgRNA RNA into the cytoplasm of mice 1 cell fertilized embryo,in which targeted gene Dmd C close to PAM,and the results showed that hyCBEs have higher efficiency for generating Dmd disease mouse models than CBEs`s.Moreover,hyeA3A-BE4 max specifically generated a C-to-T conversion without inducing bystander mutations in the hemoglobin gamma(HBG)gene promoter to mimic a naturally occurring genetic variant for amelioration of β-hemoglobinopathy.Second,we developed a dual adenine/cytosine-base editor(A&C-BEmax)by fusing both deaminases with a Cas9 nickase,which not only achieve C-to-T or A-to-G conversions,but also both C-to-T and A-to-G conversion t the same target site and more DNA mutation types.Compared to the single base editors,A&C-BEmax’s editing window and activity on adenines is slightly reduced and narrowed,respectively;whereas editing window on cytosines was expanded to 16 nucleotides(positions 2-17)compared to positions 3-13 for AID-BE4 max and editing activity on cytosines was up to 14 fold than AID-BE4max;Meanwhile,RNA off-target activity substantially decreased and low indels efficiency was maintained.By targeting the promoter HBG1/2(-113 A,-114 C,-115C)with dual base editor,different HUDEP-2 cell clones harbored C-to-T or both C-to-T and A-to-G were generated.The HUDEP-2 cell clones harbored-114C-to-T or-113 A-to-G exhibited highest γ-globin mRNA expression than others after HUDEP-2 cell clones differentiation.Our in silico analysis found 203 target sites containing known pathogenic A-to-G mutation(s)and C-to-T mutation(s)which could potentially be reverted by A&C-BEmax through a single sgRNA,albeit the SNVs may be located on different alleles.The targeting scope increased by 2.8-fold(~573)when the Cas9-NG variant was leveraged for the analysis.In summary,through our systematic engineering design for CBEs,hyCBEs breaks the technical bottleneck of CBE’s editing window and editing efficiency,greatly improves its editing efficiency and targeting range,and promotes the wide application of CBE in various fields.A&C-BEmax addressed the limitation that the single-base editor can only work on one substrate and develped a dual-function base editing platform that can use As or Cs as substrates,providing a multifunction platform for gene therapy and genome diversity.