Hoban Megan D, Lumaquin Dianne, Kuo Caroline Y, Romero Zulema, Long Joseph, Ho Michelle, Young Courtney S, Mojadidi Michelle, Fitz-Gibbon Sorel, Cooper Aaron R, Lill Georgia R, Urbinati Fabrizia, Campo-Fernandez Beatriz, Bjurstrom Carmen F, Pellegrini Matteo, Hollis Roger P, Kohn Donald B
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
Division of Allergy and Immunology, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
Mol Ther. 2016 Sep;24(9):1561-9. doi: 10.1038/mt.2016.148. Epub 2016 Jul 29.
Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.
靶向基因组编辑技术能够纠正造血干细胞中β-珠蛋白基因的镰状细胞病突变。这种纠正有助于合成正常血红蛋白蛋白的红细胞的生成。在此，我们证明转录激活样效应核酸酶（TALENs）和成簇规律间隔短回文重复序列（CRISPR）/Cas9核酸酶系统能够靶向β-珠蛋白基因中镰状细胞突变周围的DNA序列进行位点特异性切割，并且当共递送同源供体模板时能够促进精确纠正。对几对TALENs和多个CRISPR引导RNA的靶向切割率和脱靶切割率进行了评估。将CRISPR/Cas9组分递送至CD34+细胞在体外导致超过18%的基因修饰。此外，我们证明了在镰状细胞病患者来源的骨髓CD34+造血干细胞和祖细胞中镰状细胞病突变得到纠正，从而产生野生型血红蛋白。这些结果证明了使用CRISPR/Cas9技术在患者来源的CD34+细胞中纠正镰状突变。
