NHEJ-Mediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia

Roman-Rodriguez, Francisco J; Ugalde, Laura; Álvarez, Lara; Díez, Begoña; Ramı́rez, Marı́a José; Risueño, Cristina; Cortón, Marta; Bogliolo, Massimo; Bernal, Sara; March, Francesca; Ayuso, Carmen; Hanenberg, Helmut; Sevilla, Julián; Rodríguez‐Perales, Sandra; Torres-Ruíz, Raúl; Surrallés, Jordi; Bueren, Juan A.; Rı́o, Paula

doi:10.1016/j.stem.2019.08.016

Cited by 68 publications

(52 citation statements)

References 46 publications

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“…hCD34+ were obtained freshly from the umbilical cord blood of healthy donors after informed consent was obtained and after approval by the Cord Blood Bank Transfusion Center of the Community of Madrid. Mononuclear cells were purified by gradient centrifugation (Histopaque, Sigma-Aldrich) and hCD34 + cells were purified by separation using magnetic beads (Miltenyi Biotec) 54 . hCD34+ cells were cultured in StemSpan SFEM II (StemCell Technologies) containing 100 U/ mL P/S and a cytokine cocktail composed of 100 ng/mL of human stem cell factor (hSCF) human FMS-like tyrosine kinase 3 (hFlt3L) and human thrombopoietin (hTPO) and 10 ng/mL of human interleukin 3 (hIL3) (all from Preprotech).…”

Section: Methodsmentioning

confidence: 99%

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells

et al. 2020

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Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.

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Section: Methodsmentioning

confidence: 99%

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells

et al. 2020

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“…Hence, it is possible, though the chance of success is low, to achieve precise DNA modification through the NEHJ pathway. One successful example is the restoration of FANCA gene expression in haematopoietic stem cells ( 164 ). HDR efficiency is generally low (less than 2%) but, with CRISPR technology, it can be improved to 10-40%, depending on the target region ( 165 , 166 ).…”

Section: Genome Editing: Tools To Explore and Correct Genetic Defectsmentioning

confidence: 99%

Functional Genomics in Pancreatic β Cells: Recent Advances in Gene Deletion and Genome Editing Technologies for Diabetes Research

Cherkaoui

Misra

et al. 2020

Front. Endocrinol.

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The inheritance of variants that lead to coding changes in, or the mis-expression of, genes critical to pancreatic beta cell function can lead to alterations in insulin secretion and increase the risk of both type 1 and type 2 diabetes. Recently developed clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene editing tools provide a powerful means of understanding the impact of identified variants on cell function, growth, and survival and might ultimately provide a means, most likely after the transplantation of genetically “corrected” cells, of treating the disease. Here, we review some of the disease-associated genes and variants whose roles have been probed up to now. Next, we survey recent exciting developments in CRISPR/Cas9 technology and their possible exploitation for β cell functional genomics. Finally, we will provide a perspective as to how CRISPR/Cas9 technology may find clinical application in patients with diabetes.

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“…DSBs can be repaired mainly by means of non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ does not require a DNA donor, so it generates small insertions or deletions (indels) that can be also used therapeutically [1,2,[12][13][14]. On the other hand, HDR uses single-or double-stranded DNA templates with homology to the break site in order to either repair an existing mutation or to insert a new DNA fragment into a precise location [15,16].…”

Section: Introductionmentioning

confidence: 99%

Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System

Sánchez‐Hernández

Aguilar-González

Guijarro-Albaladejo

et al. 2020

Cells

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In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.

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NHEJ-Mediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia

Abstract: Highlights d NHEJ-mediated gene editing enables highly efficient editing in human long-term HSCs d NHEJ-mediated editing restores mutant coding frames across FA complementation groups d Corrected FA-HSCs have a marked proliferative advantage in vitro and in vivo

Cited by 68 publications

References 46 publications

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells

Functional Genomics in Pancreatic β Cells: Recent Advances in Gene Deletion and Genome Editing Technologies for Diabetes Research

Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System

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