RESUMEN
BACKGROUND: Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology. RESULTS: For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci. CONCLUSIONS: Our findings provide a novel efficient approach to promote precise gene editing in human cells.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Histonas/genética , Ubiquitinación , Animales , Proteína BRCA1/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteínas de Unión al ADN/genética , Expresión Génica , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Lamina Tipo B/genética , Reparación del ADN por Recombinación , Ubiquitina/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica/métodos , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Técnicas de Transferencia de Gen , ARN Guía de Kinetoplastida/genética , Animales , Animales Recién Nacidos , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Electroporación/métodos , Endonucleasas/metabolismo , Marcación de Gen/métodos , Genoma , Ratones , Ratones Transgénicos , Microinyecciones , ARN Guía de Kinetoplastida/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Reparación del ADN por Recombinación , Cigoto/citología , Cigoto/metabolismoRESUMEN
Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease mainly caused by mutations in the X-linked CYBB gene that abrogate reactive oxygen species (ROS) production in phagocytes and microbial defense. Gene repair using the CRISPR/Cas9 system in hematopoietic stem and progenitor cells (HSPCs) is a promising technology for therapy for CGD. To support the establishment of efficient and safe gene therapies for CGD, we generated a mouse model harboring a patient-derived mutation in the CYBB gene. Our CybbC517del mouse line shows the hallmarks of CGD and provides a source for Cybb-deficient HSPCs that can be used to evaluate gene-therapy approaches in vitro and in vivo. In a setup using Cas9 RNPs and an AAV repair vector in HSPCs, we show that the mutation can be repaired in 19% of treated cells and that treatment restores ROS production by macrophages. In conclusion, our CybbC517del mouse line provides a new platform for refining and evaluating novel gene therapies and studying X-CGD pathophysiology.
Asunto(s)
Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Terapia Genética , Enfermedad Granulomatosa Crónica , NADPH Oxidasa 2 , Enfermedad Granulomatosa Crónica/terapia , Enfermedad Granulomatosa Crónica/genética , Animales , Terapia Genética/métodos , Ratones , NADPH Oxidasa 2/genética , Especies Reactivas de Oxígeno/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Macrófagos/metabolismo , MutaciónRESUMEN
[This corrects the article DOI: 10.3389/fgene.2019.00365.].
RESUMEN
The CRISPR-Cas9 system is used for genome editing in mammalian cells by introducing double-strand breaks (DSBs) which are predominantly repaired via non-homologous end joining (NHEJ) or to lesser extent by homology-directed repair (HDR). To enhance HDR for improving the introduction of precise genetic modifications, we tested fusion proteins of Cas9 nuclease with HDR effectors to enforce their localization at DSBs. Using a traffic-light DSB repair reporter (TLR) system for the quantitative detection of HDR and NHEJ events in human HEK cells we found that Cas9 fusions with CtIP, Rad52, and Mre11, but not Rad51C promote HDR up to twofold in human cells and significantly reduce NHEJ events. We further compared, as an alternative to the direct fusion with Cas9, two components configurations that associate CtIP fusion proteins with a Cas9-SunTag fusion or with guide RNA that includes MS2 binding loops. We found that the Cas9-CtIP fusion and the MS2-CtIP system, but not the SunTag approach increase the ratio of HDR/NHEJ 4.5-6-fold. Optimal results are obtained by the combined use of Cas9-CtIP and MS2-CtIP, shifting the HDR/NHEJ ratio by a factor of 14.9. Thus, our findings provide a simple and effective tool to promote precise gene modifications in mammalian cells.