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

Román-Rodríguez, Francisco JoséUgalde, LauraÁlvarez, LaraDíez, BegoñaRamírez, María JoséRisueño, CristinaCortón, MartaBogliolo, MassimoBernal, SaraMarch, FrancescaAyuso, CarmenHanenberg, HelmutSevilla, JuliánRodriguez Perales, SandraTorres-Ruiz, RaulSurrallés, JordiBueren, Juan AntonioRío, Paula2025-01-272025-01-272019-11-07Cell Stem Cell . 2019 Nov 7;25(5):607-621https://hdl.handle.net/20.500.12105/26146The authors would like to thank Jonathan Schwartz for critical reading of the manuscript, Aurora de la Cal for her assistance in coordinating patients' samples, and Miguel A. Martin and Sergio Garcia for the maintenance and irradiation of the animals, respectively. This work was supported by "Ministerio de Economia, Comercio y Competitividad y Fondo Europeo de Desarrollo Regional (FEDER)" (SAF2015-68073-R, SAF2015-64152-R, and RTI2018097125-B-I00), "7th Framework Program European Commission" (HEALTHF5-2012-305421 and EUROFANCOLEN), "Ministerio de Sanidad, Servicios Sociales e Igualdad" (EC11/060 and EC11/550), "Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III" (RD12/0019/0023), ICREA-Academia program, and Rocket Pharmaceuticals Inc. R.T.-R. is supported by a postdoctoral fellowship from the Asociacion Espanola Contra el Cancer (AECC).Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-stranded DNA breaks and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here, we show feasible correction of compromised functional phenotypes in hematopoietic cells from multiple FA complementation groups, including FA-A, FA-C, FA-D1, and FA-D2. NHEJ-mediated repair of targeted CRISPR-Cas9-induced DNA breaks generated compensatory insertions and deletions that restore the coding frame of the mutated gene. NHEJ-mediated editing efficacy was initially verified in FA lymphoblastic cell lines and then in primary FA patient-derived CD34 cells, which showed marked proliferative advantage and phenotypic correction both in vitro and after transplantation. Importantly, and in contrast to homologous directed repair, NHEJ efficiently targeted primitive human HSCs, indicating that NHEJ editing approaches may constitute a sound alternative for editing self-renewing human HSCs and consequently for treatment of FA and other monogenic diseases affecting the hematopoietic system.engVoRhttp://creativecommons.org/licenses/by-nc-nd/4.0/CRISPR/Cas9 systemFanconi anemiagene editinghematopoietic stem and progenitor cellsindelsnon-homologous end-joiningNHEJ-Mediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia.Attribution-NonCommercial-NoDerivatives 4.0 International31543367255607-621Cell Stem Cellopen access