Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia.

Autor:	Ye L; Department of Medicine, University of California, San Francisco, CA 94143; Institute for Human Genetics, University of California, San Francisco, CA 94143; yw.kan@ucsf.edu lin.ye@ucsf.edu., Wang J; Department of Medicine, University of California, San Francisco, CA 94143; Institute for Human Genetics, University of California, San Francisco, CA 94143; Department of Laboratory Medicine, University of California, San Francisco, CA 94143;, Tan Y; Department of Medicine, University of California, San Francisco, CA 94143; State Key Laboratory of Medical Genomics, Shanghai Jiao Tong University, Shanghai 200025, China; Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China;, Beyer AI; Blood Systems Research Institute, San Francisco, CA 94118., Xie F; Department of Medicine, University of California, San Francisco, CA 94143; Institute for Human Genetics, University of California, San Francisco, CA 94143;, Muench MO; Department of Laboratory Medicine, University of California, San Francisco, CA 94143; Blood Systems Research Institute, San Francisco, CA 94118., Kan YW; Department of Medicine, University of California, San Francisco, CA 94143; Institute for Human Genetics, University of California, San Francisco, CA 94143; Department of Laboratory Medicine, University of California, San Francisco, CA 94143; yw.kan@ucsf.edu lin.ye@ucsf.edu.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2016 Sep 20; Vol. 113 (38), pp. 10661-5. Date of Electronic Publication: 2016 Sep 06.
DOI:	10.1073/pnas.1612075113
Abstrakt:	Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher γ-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous β-thalassemia and SCD. Competing Interests: The authors declare no conflict of interest.
Databáze:	MEDLINE
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