Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Wienert, Beeke; Nguyen, David N.; Guenther, Alexis; Feng, Sharon; Locke, Melissa N.; Wyman, Stacia K.; Shin, Jiyung; Kazane, Katelynn R.; Gregory, Georgia L.; Carter, Matthew; Wright, Francis; Conklin, Bruce R.; Marson, Alex; Richardson, Christopher D.; Corn, Jacob E.

doi:10.1038/s41467-020-15845-1

Cited by 95 publications

(80 citation statements)

References 58 publications

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“…KI rates were doubled compared to embryos not treated with RS-1 [61]. Another recent study, however, did not observe an effect with RS-1 [41].…”

Section: Rad51mentioning

confidence: 81%

“…They are shown with their functional target and observed effects in cell lines and animal models. It is further distinguished between single strand (ss) and double-strand (ds) donors, as it has been shown that repair by single-strand donors involves a different set of repair factors than repair by double-strand donors [40,41].…”

Section: Small Molecules To Improve Precise Genome Editingmentioning

confidence: 99%

“…The Corn group performed a screen to identify regulators of HDR with a double-stranded DNA repair template. Using a reporter system for HDR driven conversion of Blue Fluorescent Protein (BFP) to Green Fluorescent Protein (GFP) expressed in K562 cells, they identified CDC7 as a repressor of HDR [41]. Since XL413 had previously been shown to specifically inhibit CDC7 [74] they then investigated whether this inhibitor might increase HDR efficiency.…”

Section: Increase Of Homology Directed Repair By Cell Cycle Synchronimentioning

confidence: 99%

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Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

Bischoff

Wimberger

Maresca

et al. 2020

Cells

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome.

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“…KI rates were doubled compared to embryos not treated with RS-1 [61]. Another recent study, however, did not observe an effect with RS-1 [41].…”

Section: Rad51mentioning

confidence: 81%

Section: Small Molecules To Improve Precise Genome Editingmentioning

confidence: 99%

Section: Increase Of Homology Directed Repair By Cell Cycle Synchronimentioning

confidence: 99%

See 1 more Smart Citation

Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

Bischoff

Wimberger

Maresca

et al. 2020

Cells

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“…At least one company plans to test these inhibitors in the context of clinical genome engineering (23). Some of the alternative strategies include modulation of the cell cycle and of the HR pathway itself (24)(25)(26)(27)(28).…”

Section: Introductionmentioning

confidence: 99%

Cas9-induced large deletions and small indels are controlled in a convergent fashion

Kosicki

Allen

Bradley

2020

Preprint

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Repair of Cas9-induced double-stranded breaks results primarily in formation of small indels, but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a novel library of clonal mouse embryonic stem cells bona fide deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a novel role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.

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“…Nonetheless, it will be important to monitor such parameters during clinical development. It will also be interesting to investigate approaches to reduce the formation of these loss-of-function mutations during the gene editing process 43–45 and also limit the toxicity of gene editing on HSCs 10 .…”

Section: Discussionmentioning

confidence: 99%

Cas9-AAV6 Gene Correction of Beta-Globin in Autologous HSCs Improves Sickle Cell Disease Erythropoiesis in Mice

Wilkinson

Dever

Baik

et al. 2020

Preprint

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CRISPR/Cas9-mediated beta-globin (HBB) gene correction of Sickle Cell Disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a novel paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated. Here, we used a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observed a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

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Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Cited by 95 publications

References 58 publications

Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

Cas9-induced large deletions and small indels are controlled in a convergent fashion

Cas9-AAV6 Gene Correction of Beta-Globin in Autologous HSCs Improves Sickle Cell Disease Erythropoiesis in Mice

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