Assuntos
Anemia de Fanconi/terapia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação A da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Condicionamento Pré-Transplante/métodosRESUMO
The efficient site-specific DNA double-strand breaks (DSB) created by CRISPR/Cas9 has revolutionized genome engineering and has great potential for editing hematopoietic stem/progenitor cells (HSPCs). However, detailed understanding of the variables that influence choice of DNA-DSB repair (DDR) pathways by HSPC is required for therapeutic levels of editing in these clinically relevant cells. We developed a hematopoietic-reporter system that rapidly quantifies the three major DDR pathways utilized at the individual DSB created by CRISPR/Cas9-NHEJ, MMEJ, and HDR-and show its applicability in evaluating the different DDR outcomes utilized by human hematopoietic cell lines and primary human HSPC.
Assuntos
Sistemas CRISPR-Cas/genética , Reparo do DNA/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/citologia , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Reparo do DNA , Edição de Genes/métodos , Reparo de DNA por Recombinação/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Humanos , Mutagênese Insercional , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
Sickle cell disease is the most prevalent monogenic disorder worldwide and curative therapies are limited to hematopoietic stem cell transplant to the few with matched donors. Gene therapy has curative potential, whereby autologous hematopoietic stem cells are genetically modified and transplanted, which would not be limited by matched donors, resulting in 1-time, life-long correction devoid of immune side effects. Significant progress has been made to clinically translate gene therapy for sickle cell disease using lentivirus vectors carrying antisickling genes. This review focuses on the current state of the field, factors that determine clinical success, gene editing, and future prospects.