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Badran, Ahmed

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Badran

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Ahmed

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Badran, Ahmed
Author's Publications: search.filters.isAuthorOfPublication.572c278f-f485-4cc5-8676-d49cc5c358b1

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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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    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.
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    Negative selection and stringency modulation in phage-assisted constinuous evolution
    (2014) Carlson, Jacob Charles; Badran, Ahmed; Guggiana-Nilo, Drago A.; Liu, David
    Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to dramatically accelerate laboratory evolution experiments. In this work we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule-controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counter-selection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ∼10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates, while maintaining wild-type-like levels of activity.
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    Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    (2016) Badran, Ahmed; Guzov, Victor M.; Huai, Qing; Kemp, Melissa M.; Vishwanath, Prashanth; Kain, Wendy; Nance, Autumn M.; Evdokimov, Artem; Moshiri, Farhad; Turner, Keith H.; Wang, Ping; Malvar, Thomas; Liu, David
    The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. We developed a phage-assisted continuous evolution (PACE) selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.
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    Continuous directed evolution of DNA-binding proteins to improve TALEN specificity
    (2015) Hubbard, Basil P.; Badran, Ahmed; Zuris, John A.; Guilinger, John P.; Davis, Kevin; Chen, Liwei; Tsai, Shengdar Q.; Sander, Jeffry D.; Joung, J. Keith; Liu, David
    Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate TALE nucleases with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents.
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    Development of potent in vivo mutagenesis plasmids with broad mutational spectra
    (Nature Pub. Group, 2015) Badran, Ahmed; Liu, David
    Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms.
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    Advances in Phage-Assisted Continuous Evolution and Application to Overcoming Bioinsecticide Resistance
    (2016-05-20) Badran, Ahmed; Liu, David R.; Kahne, Daniel; Hochschild, Ann; Balskus, Emily P.
    The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits, constituting a substantial and increasingly large portion of the total global production of various crops, including cotton, corn, maize and soybeans. Bt toxins are exquisitely selective for targeted pests, typically do not affect off-target insects, and are completely orthogonal to human biology. However, the development of insect resistance to Bt toxins endangers their long-term effectiveness. In this thesis, I describe the development of methodology for the systematic directed evolution of novel Bt toxins to selectively affect resistant insects. Using Phage-Assisted Continuous Evolution (PACE), a previously developed general platform for the continuous directed evolution of biomolecules, I developed a highly sensitized selection for novel protein-protein interactions. This system robustly reported on interactions spanning affinities from low micromolar to picomolar. However, attempts at using this system for the directed evolution of novel protein-protein interactions were largely unsuccessful, presumably as a consequence of low mutagenesis efficiency. To increase the utility of the platform, I sought to enhance the mutagenesis levels afforded by PACE, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency, and narrow mutational spectra. I used a systematic, mechanism-driven approach to create a potent, inducible, broad-spectrum, and vector-based mutagenesis system in E. coli that enhances mutation rates by 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro mutagenesis methods. This system enabled the high-frequency, broad-spectrum mutagenesis of chromosomal, episomal, and viral nucleic acids in vivo, and dramatically enhanced the success of PACE experiments, highlighting the importance of mutagenesis efficiency on selection outcome. Using this enhanced mutagenesis approach and the previously described sensitized selection platform, I was able to evolve variants of the commonly used Bt toxin Cry1Ac that bind toxin binding region of a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. In conclusion, these finding offer a novel mechanism of overcoming what is quickly becoming among the largest issue overshadowing the continued success of Bt toxins for pest control and management, and establish a platform for the detection and evolution of a wide array of protein-protein interactions.
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    In vivo continuous directed evolution
    (Elsevier BV, 2015) Badran, Ahmed; Liu, David
    The development and application of methods for the laboratory evolution of biomolecules has rapidly progressed over the last few decades. Advancements in continuous microbe culturing and selection design have facilitated the development of new technologies that enable the continuous directed evolution of proteins and nucleic acids. These technologies have the potential to support the extremely rapid evolution of biomolecules with tailor-made functional properties. Continuous evolution methods must support all of the key steps of laboratory evolution—translation of genes into gene products, selection or screening, replication of genes encoding the most fit gene products, and mutation of surviving genes—in a self-sustaining manner that requires little or no researcher intervention. Continuous laboratory evolution has been historically used to study problems including antibiotic resistance, organismal adaptation, phylogenetic reconstruction, and host-pathogen interactions, with more recent applications focusing on the rapid generation of proteins and nucleic acids with useful, tailor-made properties. The advent of increasingly general methods for continuous directed evolution should enable researchers to address increasingly complex questions and to access biomolecules with more novel or even unprecedented properties.