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Publication Record


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 AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, Kim YB, Badran AH, Liu DR
(2017) Sci Adv 3: eaao4774
MeSH Terms: Bacteriophage mu, Base Pairing, Cell Line, DNA Repair, DNA-Binding Proteins, Enzyme Activation, Gene Frequency, Gene Order, Humans, INDEL Mutation, Uracil-DNA Glycosidase, Viral Proteins
Show Abstract · Added March 21, 2018
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.
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12 MeSH Terms
Roles of fimB and fimE in site-specific DNA inversion associated with phase variation of type 1 fimbriae in Escherichia coli.
McClain MS, Blomfield IC, Eisenstein BI
(1991) J Bacteriol 173: 5308-14
MeSH Terms: Bacteriophage mu, Base Sequence, Chromosome Inversion, DNA, Bacterial, DNA, Recombinant, Electrophoresis, Agar Gel, Escherichia coli, Fimbriae, Bacterial, Genes, Viral, Molecular Sequence Data, Mutation, Plasmids, Restriction Mapping
Show Abstract · Added May 20, 2014
Evidence obtained with an improved in vivo assay of fimbrial phase variation in Escherichia coli supported a revised understanding of the roles of fimB and fimE in the site-specific DNA rearrangement with which they are associated. A previously proposed model argued that fimB and fimE play antagonistic, unidirectional roles in regulating the orientation of the invertible DNA element located immediately upstream of fimA, the gene encoding the major subunit of type 1 fimbriae. This conclusion, though, is based on an in vivo DNA inversion assay using recombinant plasmid substrates under conditions that, among other things, were incapable of detecting recombination of the fim invertible element from the on to the off orientation. Using a modified system that overcome this and several additional technical problems, we confirmed that fimB acts independently of fimE on the invertible element and that the additional presence of fimE results in the preferential rearrangement of the element to the off orientation. It is now demonstrated that fimE can act in the absence of fimB in this recombination to promote inversion primarily from on to off. In contrast to the previous studies, the effect of fimB on a substrate carrying the invertible element in the on orientation could be examined. It was found that fimB mediates DNA inversion from on to off, as well as from off to on, and that, contrary to prior interpretations, the fimB-associated inversion occurs with only minimal orientational preference to the on phase.
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13 MeSH Terms