High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing

• Published in: Molecular therapy. Nucleic acids 2018-03, Vol.10, p.1-8
• Main Authors: Sürün, Duran, Schwäble, Joachim, Tomasovic, Ana, Ehling, Roy, Stein, Stefan, Kurrle, Nina, von Melchner, Harald, Schnütgen, Frank
• Format: Article
• Language: English
• Subjects: Amino acids; CGD; Chronic granulomatous disease; Cloning; CRISPR; CRISPR/Cas9; Cytochrome b; Deoxyribonucleic acid; DNA; Efficiency; Frameshift mutation; Gene therapy; Genome editing; Genomes; Hematological diseases; hematopoietic cells; Immune system; Immunodeficiency; in situ gene correction; Infections; Leukocytes (neutrophilic); Macrophages; Microbicides; Mutagenesis; Mutation; NHEJ; non-homologous end joining; Nucleotide sequence; Proteins; Reactive oxygen species; Ribonucleic acid; RNA; Stem cells; Vectors (Biology)
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2017.11.001

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs. [Display omitted]