Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity
Komor, Alexis C.
Packer, Michael S.
Kim, Y. Bill
MetadataShow full item record
CitationKomor, Alexis C., Kevin T. Zhao, Michael S. Packer, Nicole M. Gaudelli, Amanda L. Waterbury, Luke W. Koblan, Y. Bill Kim, Ahmed H. Badran, and David R. Liu. 2017. “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity.” Science Advances 3 (8): eaao4774. doi:10.1126/sciadv.aao4774. http://dx.doi.org/10.1126/sciadv.aao4774.
AbstractWe recently developed base editing, the programmable conversion of target C:G base pairs to T:A without inducing double-stranded DNA breaks (DSBs) or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases. Over the past year, the third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms. The product distribution of base editing—the frequency with which the target C:G is converted to mixtures of undesired by-products, along with the desired T:A product—varies in a target site–dependent manner. We characterize determinants of base editing outcomes in human cells and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG) and is more likely to occur at target sites containing only a single C within the base editing activity window. We engineered CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences. On the basis of these observations, we engineered fourth-generation base editors (BE4 and SaBE4) that increase the efficiency of C:G to T:A base editing by approximately 50%, while halving the frequency of undesired by-products compared to BE3. Fusing BE3, BE4, SaBE3, or SaBE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing, in most cases to below 1.5%, and further improves product purity. BE4, SaBE4, BE4-Gam, and SaBE4-Gam represent the state of the art in C:G-to-T:A base editing, and we recommend their use in future efforts.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:34492081