Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity

By Alexis C. Komor, K. Zhao, M. Packer, Nicole M. Gaudelli, Amanda L. Waterbury, Luke W. Koblan, Y. B. Kim, A. Badran, David R. Liu. 2017

708 citations recorded by the source corpus.

Probing base editing outcomes leads to new C:G to T:A base editors with greater efficiency and product purity, and fewer indels. We 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.

Subfields: Genetics, Molecular Biology

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Citations in the corpus

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, positive): “We also tested xCas9 3.7 in the BE4 architecture designed to reduce undesired byproducts 34 , and observed fewer indels and higher product purities, although with slightly lower editing efficiencies (Extended Data Fig.”
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage (2017, neutral): “…a uracil glycosylase inhibitor (UGI) that impedes uracil excision and downstream processes that decrease base editing efficiency and product purity 5 , and (iv) nickase activity to nick the non-edited DNA strand, directing cellular DNA repair processes to replace the G-containing DNA strand 3,5 .”
CRISPR technology: A decade of genome editing is only the beginning (2023, background): “It has been demonstrated that fusing base editors to Gam—a bacteriophage Mu protein that binds DSBs—can minimize indel formation during base editing (129).”
Adeno-associated virus as a delivery vector for gene therapy of human diseases (2024, background): “146,147 Various strategies have emerged to overcome this hurdle by dividing a large transgene into two parts, with each encapsulated in an rAAV capsid.”