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Koblan, Luke

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Koblan

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Luke

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Koblan, Luke
Author's Publications: search.filters.isAuthorOfPublication.5fdb1515-2081-432a-ac9b-967373e352ca

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Now showing 1 - 7 of 7
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    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
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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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    Search-and-Replace Genome Editing Without Double-Strand Breaks or Donor DNA
    (Springer Science and Business Media LLC, 2019-10-21) Anzalone, Andrew; Randolph, Peyton B.; Davis, Jessie R.; Sousa, Alexander; Koblan, Luke; Levy, Jonathan; Chen, Peter; Wilson, Christopher; Newby, Gregory; Raguram, Aditya; Liu, David R.
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    Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning
    (Springer Science and Business Media LLC, 2021-06-28) Koblan, Luke; Arbab, Mandana; Shen, Max; Hussmann, Jeffrey A.; Anzalone, Andrew; Doman, Jordan; Newby, Gregory; Yang, Dian; Mok, Beverly; Replogle, Joseph M.; Xu, Albert; Sisley, Tyler A.; Weissman, Jonathan S.; Adamson, Brittany; Liu, David
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    Continuous Evolution of Base Editors With Expanded Target Compatibility and Improved Activity
    (Springer Science and Business Media LLC, 2019-07-22) Zheng, Christine; Wilson, Christopher; Thuronyi, Benjamin; Koblan, Luke; Levy, Jonathan; Yeh, Wei-Hsi; Newby, Gregory; Bhaumik, Mantu; Shubina-Oleinik, Olga; Holt, Jeffrey; Liu, David
    Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE–PACE) to improve their editing efficiency and target sequence compatibility. We used BE–PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.
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    Adenine Base Editing in an Adult Mouse Model of Tyrosinaemia
    (Springer Science and Business Media LLC, 2019-02-25) Song, Chun-Qing; Jiang, Tingting; Richter, Michelle; Rhym, Luke H.; Koblan, Luke; Paz Zafra, Maria; Schatoff, Emma M.; Doman, Jordan; Cao, Yueying; Dow, Lukas E.; Zhu, Lihua Julie; Anderson, Daniel; Liu, David; Yin, Hao; Xue, Wen
    Unlike traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here, we show in a mouse model of tyrosinemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single guide RNA can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes in the liver, and rescued weight loss in the animals. We also generated Fah+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABE. Our findings suggest that adenosine base editing can be used for the correction of genetic disease in adult animals.
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    Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing
    (Springer Science and Business Media LLC, 2021-12-09) Anzalone, Andrew; Gao, Xin; Podracky, Christopher J.; Nelson, Andrew; Koblan, Luke; Raguram, Aditya; Levy, Jonathan; Mercer, Jaron; Liu, David
    The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.