Person:
Zhao, Kevin

Profile Picture

Email Address

AA Acceptance Date

Birth Date

Research Projects

Organizational Units

Job Title

Last Name

Zhao

First Name

Kevin

Name

Zhao, Kevin
Author's Publications: search.filters.isAuthorOfPublication.0dd5a2c9-40c6-4529-8cb9-e487e15178d1

Search Results

Now showing 1 - 5 of 5
  • No Thumbnail Available
    Publication
    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.
  • No Thumbnail Available
    Publication
    Reconstruction of evolving gene variants and fitness from short sequencing reads
    (Springer Science and Business Media LLC, 2021-10-11) Shen, Max; Zhao, Kevin; Liu, David
    Directed evolution can generate proteins with tailor-made activities. However, full-length genotypes, their frequencies and fitnesses are difficult to measure for evolving gene-length biomolecules using most high-throughput DNA sequencing methods, as short read lengths can lose mutation linkages in haplotypes. Here we present Evoracle, a machine learning method that accurately reconstructs full-length genotypes (R2 = 0.94) and fitness using short-read data from directed evolution experiments, with substantial improvements over related methods. We validate Evoracle on phage-assisted continuous evolution (PACE) and phage-assisted non-continuous evolution (PANCE) of adenine base editors and OrthoRep evolution of drug-resistant enzymes. Evoracle retains strong performance (R2 = 0.86) on data with complete linkage loss between neighboring nucleotides and large measurement noise, such as pooled Sanger sequencing data (~US$10 per timepoint), and broadens the accessibility of training machine learning models on gene variant fitnesses. Evoracle can also identify high-fitness variants, including low-frequency ‘rising stars’, well before they are identifiable from consensus mutations
  • No Thumbnail Available
    Publication
    Base editing of haematopoietic stem cells rescues sickle cell disease in mice
    (Springer Science and Business Media LLC, 2021-06-02) Newby, Gregory; Yen, Jonathan S.; Woodard, Kaitly J.; Mayuranathan, Thiyagaraj; Lazzarotto, Cicera R.; Li, Yichao; Sheppard-Tillman, Heather; Porter, Shaina N.; Yao, Yu; Mayberry, Kalin; Everette, Kelcee A.; Jang, Yoonjeong; Podracky, Christopher J.; Thaman, Elizabeth; Lechauve, Christophe; Sharma, Akshay; Henderson, Jordana M.; Richter, Michelle; Zhao, Kevin; Miller, Shannon; Wang, Tina; Koblan, Luke; McCaffrey, Anton P.; Tisdale, John F.; Kalfa, Theodosia; Pruett-Miller, Shondra M.; Tsai, Shengdar; Weiss, Mitchell J.; Liu, David
  • Thumbnail Image
    Publication
    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.
  • Thumbnail Image
    Publication
    Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions
    (2017) Kim, Y. Bill; Komor, Alexis C.; Levy, Jonathan; Packer, Michael S.; Zhao, Kevin; Liu, David
    Base editing is a recently developed approach to genome editing that uses a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair to induce programmable, single-nucleotide changes in the DNA of living cells without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions1. Here we report the development of five new C→T (or G→A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing by 2.5-fold. Additionally, we engineered new base editors containing mutated cytidine deaminase domains that narrow the width of the apparent editing window from approximately 5 nucleotides to as little as 1 to 2 nucleotides, enabling the discrimination of neighboring C nucleotides that would previously be edited with comparable efficiency, thereby doubling the number of disease-associated target Cs that can be corrected preferentially over nearby non-target Cs. Collectively, these developments substantially increase the targeting scope of base editing and establish the modular nature of base editors.