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Gaudelli, Nicole

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Gaudelli

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Nicole

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Gaudelli, Nicole
Author's Publications: search.filters.isAuthorOfPublication.29a41bc5-1ebf-4328-8037-41e69b2ba439

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    Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors
    (Springer Science and Business Media LLC, 2019-05-20) Huang, Tony; Zhao, Kevin; Miller, Shannon; Gaudelli, Nicole; Oakes, Benjamin; Fellmann, Christof; Savage, David; Liu, David
    Base editing requires that the target sequence satisfy the protospacer adjacent motif requirement of the Cas9 domain and that the target nucleotide be located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG protospacer adjacent motifs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4–5 nucleotides to up to ~8–9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.
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    Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity
    (American Association for the Advancement of Science, 2017) Komor, Alexis C.; Zhao, Kevin; Packer, Michael S.; Gaudelli, Nicole; Waterbury, Amanda; Koblan, Luke; Kim, Y. Bill; Badran, Ahmed; Liu, David
    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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    Publication
    Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    (2017) Gaudelli, Nicole; Komor, Alexis C.; Rees, Holly; Packer, Michael S.; Badran, Ahmed; Bryson, David I.; Liu, David
    Summary The spontaneous deamination of cytosine is a major source of C•G to T•A transitions, which account for half of known human pathogenic point mutations. The ability to efficiently convert target A•T base pairs to G•C therefore could advance the study and treatment of genetic diseases. While the deamination of adenine yields inosine, which is treated as guanine by polymerases, no enzymes are known to deaminate adenine in DNA. Here we report adenine base editors (ABEs) that mediate conversion of A•T to G•C in genomic DNA. We evolved a tRNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs (e.g., ABE7.10), that convert target A•T to G•C base pairs efficiently (~50% in human cells) with very high product purity (typically ≥ 99.9%) and very low rates of indels (typically ≤ 0.1%). ABEs introduce point mutations more efficiently and cleanly than a current Cas9 nuclease-based method, induce less off-target genome modification than Cas9, and can install disease-correcting or disease-suppressing mutations in human cells. Together with our previous base editors, ABEs advance genome editing by enabling the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.