Development Of An Enhanced Effective Cytosine Base Editor And Its Application In Hematopoietic Stem Cells

Base editing technology is a new genome modification method based on CRISPR/Cas system and base deaminase,which mainly includes: cytosine base editor(CBE)and adenine base editor(ABE).Cytosine base editor(CBE)can catalyze the deamination of cytosine(C)and result in uracil(U)through catalytically disabled Cas nuclease carrying deaminase,thereby generating a C to thymine(T)base conversion.CBE not only could introduce or repair the pathogenic mutation,but also achieve targeted gene knockout by generating a stop codon,which has been widely used in animal model construction and disease treatment.However,the base editing efficiency of CBE varies considerably with different genomic sites due to the complexity of chromatin structures of eukaryotic genomic DNA.In addition,as an effective tool for disease treatment,CBE shows encouraging application potential in hematopoietic stem cells,while the expression and purification of high-quality CBE protein and efficient base editing in hematopoietic stem cells are the key factors to restrict its application.Therefore,we hope to build an efficiency enhanced CBE(ee CBE)tools by enhancing the binding ability of Cas9 nickase(Cas9n)nuclease,and express and purify the CBE protein to achieve efficient base editing in the therapeutic target of hematopoietic stem cells.Firstly,we fused the DNA double stranded domain into Cas9 n nuclease to enhance the DNA binding ability,and lead to improvement of C-to-T editing efficiency.Accordingly,we developed an ee BE4 max tool to enhance base editing efficiency up to1.3-fold,while the editing window,product purity and indels rates have no obvious change.In order to expand the targeting scope of ee BE4 max,we replaced Sp Cas9 n nuclease with other Sp Cas9 n variants,and constructed a series of ee BE4 max variants targeting "NGN" PAM and near-PAMless.We also replaced the deaminase of ee BE4 max with more active APOBEC3 A to generate the most efficient ee A3ABE4 max in our study,and ee A3A-BE4 max can achieve up to 80% C to T conversion rates.Then,we established a prokaryotic expression system for CBE protein,and purified high quality A3A-BE3 and ee A3A-BE3 proteins by nickel column and molecular sieve column.Finally,we electroporated ee CBE and sg RNA as RNP complex into hematopoietic stem cells to edit the C base of BCL11 A enhancer region and HBG1/2 promoter region,results show that ee A3A-BE3 improve base editing efficiency significantly,and C to T conversion rates up to 70% at BCL11 A site,which reactivated ?-globin expression levels up to 50% relative to ?-globin levels.We also detected the off-target effect caused by ee A3A-BE3 was no significant difference when compared with A3A-BE3.Overall,we developed a variety of efficiency enhanced CBE tools by improving the binding ability of Cas9 n nuclease.We also purified the high quality ee A3A-BE3 protein to generate C to T conversion and reactivate ?-globin expression in hematopoietic stem cells,which laid a theoretical and technical foundation for treatment of ?-thalassemia disease.