Rapid genome editing by CRISPR-Cas9-POLD3 fusion

Reint, Ganna; Li, Zhuokun; Labun, Kornel; Keskitalo, Salla; Soppa, Inkeri; Mamia, Katariina; Tölö, Eero; Szymańska, Monika; Meza‐Zepeda, Leonardo A.; Lorenz, Susanne; Cieślar-Pobuda, Artur; Hu, Xian; Bordin, Diana Lilian; Staerk, Judith; Valen, Eivind; Schmierer, Bernhard; Varjosalo, Markku; Taipale, Jussi; Haapaniemi, Emma

doi:10.7554/elife.75415

Cited by 14 publications

(11 citation statements)

References 44 publications

(94 reference statements)

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“…In fact, certain strategies that have been employed to improve HDR-mediated editing by DSB-reliant tools may be effective at improving editing efficiencies by dCas9-derived BEs. These include fusion of Geminin to the editor to enhance its expression levels during S-phase ( Gutschner et al, 2016 ), or fusion of DNA Polymerase D3 (which is involved in genome replication) to the editor ( Reint et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

Examination of the Cell Cycle Dependence of Cytosine and Adenine Base Editors

et al. 2022

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Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Due to their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs rely on more ubiquitous DNA repair pathways than DSB-reliant genome editing methods, which require processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing using cell synchronization experiments. We find that nickase-derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S-phase (DNA synthesis phase). We found that synchronization in G1 (growth phase) during the process of cytosine base editing causes significant increases in C•G to A•T “byproduct” introduction rates, which can be leveraged to discover new strategies for precise C•G to A•T base editing. We observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes, and the process of base editing does not significantly perturb transcription levels. Overall, our study provides mechanistic data demonstrating the robustness of nickase-derived BEs for performing genome editing across the cell cycle.

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Section: Discussionmentioning

confidence: 99%

Examination of the Cell Cycle Dependence of Cytosine and Adenine Base Editors

et al. 2022

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“…In 2021, De Ravin and others showed that i53, an engineered ubiquitin variant with a high affinity to the tandem Tudor domain of 52BP1, effectively reduces NHEJ repair, and promotes HDR in CD34 + HSPCs ( De Ravin et al, 2021 ). Finally, other proteins tested in non-HSPCs cellular models showed great potential in increasing HDR efficiency, such as CtIP ( Charpentier et al, 2018 ), AUNIP ( Lou et al, 2017 ) or Pold-3 ( Reint et al, 2021 ).…”

Section: Methods To Improve Hdr-mediated Gene Editing In Hspcsmentioning

confidence: 99%

Hematopoietic stem and progenitors cells gene editing: Beyond blood disorders

et al. 2023

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Lessons learned from decades-long practice in the transplantation of hematopoietic stem and progenitor cells (HSPCs) to treat severe inherited disorders or cancer, have set the stage for the current ex vivo gene therapies using autologous gene-modified hematopoietic stem and progenitor cells that have treated so far, hundreds of patients with monogenic disorders. With increased knowledge of hematopoietic stem and progenitor cell biology, improved modalities for patient conditioning and with the emergence of new gene editing technologies, a new era of hematopoietic stem and progenitor cell-based gene therapies is poised to emerge. Gene editing has the potential to restore physiological expression of a mutated gene, or to insert a functional gene in a precise locus with reduced off-target activity and toxicity. Advances in patient conditioning has reduced treatment toxicities and may improve the engraftment of gene-modified cells and specific progeny. Thanks to these improvements, new potential treatments of various blood- or immune disorders as well as other inherited diseases will continue to emerge. In the present review, the most recent advances in hematopoietic stem and progenitor cell gene editing will be reported, with a focus on how this approach could be a promising solution to treat non-blood-related inherited disorders and the mechanisms behind the therapeutic actions discussed.

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“…screened the fusion of 450 DNA repair proteins with Cas9 for those that increase HDR in HEK293T green fluorescent protein (GFP) reporter cells. 74 They found approximately 31 fusions improved HDR, with many belonging to the replication fork machinery, including several replicative polymerases. They further studied the seven top proteins and found that the fusion performance was strongly dependent on individual loci and cell types, with POLD3 outperforming the other fusions, increasing HDR by ∼2 fold.…”

Section: Attempts To Increase Hdr Levelsmentioning

confidence: 99%