A marker-free co-selection strategy for high efficiency human genome engineering

Agudelo, Daniel; Bozoyan, Lusiné; Duringer, Alexis; Huard, Caroline; Carter, S; Loehr, Jérémy; Synodinou, Dafni; Drouin, Mathieu; Salsman, Jayme; Dellaire, Graham; Laganière, Josée; Doyon, Yannick

doi:10.1101/116251

Cited by 4 publications

(3 citation statements)

References 43 publications

(23 reference statements)

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“…Dever and others were able to achieve 90% HBB-edited HSPCs in long-term graft by embedding a reporter cassette in the HDR template (Dever et al, 2016). Another research group designed a robust co-selection strategy using CRISPR-Cas9 and CRISPR-Cpf1 to generate dominant cellular resistance to ouabain and simultaneously edit a second locus of interest (Agudelo et al, 2017). Another possibility to improve editing frequency in HSPCs is the exploitation of the NHEJ pathway to bypass the inefficiencies of HDRmediated approaches (Bloomer et al, 2021) (Roman-Rodriguez et al, 2019.…”

Section: Additional Strategies: Selection Of Corrected Cells and Bypa...mentioning

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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Section: Additional Strategies: Selection Of Corrected Cells and Bypa...mentioning

confidence: 99%

Hematopoietic stem and progenitors cells gene editing: Beyond blood disorders

et al. 2023

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“…Such templates can repair DSB using HR, MMEJ and SSA mechanisms, depending on the homology arm's length. They are usually used to insert long fragments (>1 kb) and make it possible to encode several required sequences and obtain biallelic gene insertions as well as multiplex 2x/3x gene modifications (Agudelo et al, 2017;Barylski et al, 2020;Branton et al, 2020;Zhang et al, 2022).…”

Section: Linear Long Dsdnamentioning

confidence: 99%

In search of an ideal template for therapeutic genome editing: A review of current developments for structure optimization

Shakirova

Karpov²,

Komarova³

et al. 2023

Front. Genome Ed.

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Gene therapy is a fast developing field of medicine with hundreds of ongoing early-stage clinical trials and numerous preclinical studies. Genome editing (GE) now is an increasingly important technology for achieving stable therapeutic effect in gene correction, with hematopoietic cells representing a key target cell population for developing novel treatments for a number of hereditary diseases, infections and cancer. By introducing a double strand break (DSB) in the defined locus of genomic DNA, GE tools allow to knockout the desired gene or to knock-in the therapeutic gene if provided with an appropriate repair template. Currently, the efficiency of methods for GE-mediated knock-in is limited. Significant efforts were focused on improving the parameters and interaction of GE nuclease proteins. However, emerging data suggests that optimal characteristics of repair templates may play an important role in the knock-in mechanisms. While viral vectors with notable example of AAVs as a donor template carrier remain the mainstay in many preclinical trials, non-viral templates, including plasmid and linear dsDNA, long ssDNA templates, single and double-stranded ODNs, represent a promising alternative. Furthermore, tuning of editing conditions for the chosen template as well as its structure, length, sequence optimization, homology arm (HA) modifications may have paramount importance for achieving highly efficient knock-in with favorable safety profile. This review outlines the current developments in optimization of templates for the GE mediated therapeutic gene correction.

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“…This protocol describes a marker-free co-selection strategy for high efficiency homology-driven and NHEJ-based gene editing in human cells by generating dominant cellular resistance to ouabain, a highly potent plant-derived inhibitor of the Na + ,K + -ATPase (1,2,3). This scarless coconversion strategy is highly efficient and can yield a stable population of modified cells in 14 days.…”

Section: Introductionmentioning

confidence: 99%

A marker-free co-selection strategy for high efficiency homology-driven and NHEJ-based gene editing in human cells

Agudelo¹,

Doyon²,

Duringer³

et al. 2017

Protocol Exchange

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Genome editing using designer nucleases has revolutionized molecular biology by allowing DNA to be inserted, deleted or replaced in the genome of several model organisms. This protocol describes a marker-free co-selection strategy for high efficiency homology-driven and NHEJ-based gene editing in human cells by generating dominant cellular resistance to ouabain, a highly potent plant-derived inhibitor of the Na+,K+ ATPase (1, 2, 3). This scarless coconversion strategy is highly efficient and can yield a stable population of modified cells in 14 days. Techniques relative to sgRNA cloning, cell nucleofection, selection using ouabain and validation of gene disruption/insertion are described.

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A marker-free co-selection strategy for high efficiency human genome engineering

Cited by 4 publications

References 43 publications

Hematopoietic stem and progenitors cells gene editing: Beyond blood disorders

Hematopoietic stem and progenitors cells gene editing: Beyond blood disorders

In search of an ideal template for therapeutic genome editing: A review of current developments for structure optimization

A marker-free co-selection strategy for high efficiency homology-driven and NHEJ-based gene editing in human cells

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