Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia.
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.
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.
Lin
L
Ye
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;
Jiaming
J
Wang
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;
Yuting
Y
Tan
Blood Systems Research Institute, San Francisco, CA 94118.
Ashley I
AI
Beyer
Department of Medicine, University of California, San Francisco, CA 94143; Institute for Human Genetics, University of California, San Francisco, CA 94143;
Fei
F
Xie
Department of Laboratory Medicine, University of California, San Francisco, CA 94143; Blood Systems Research Institute, San Francisco, CA 94118.
Marcus O
MO
Muench
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.
Yuet Wai
YW
Kan
D000755:Anemia, Sickle Cell / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D064113:CRISPR-Cas Systems; D059766:DNA End-Joining Repair / Q000235:genetics; D005319:Fetal Hemoglobin / Q000235:genetics* / Q000627:therapeutic use; D000072669:Gene Editing / Q000379:methods*; D015316:Genetic Therapy; D015894:Genome, Human; D005838:Genotype; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006579:Heterozygote; D006801:Humans; D017384:Sequence Deletion; D017086:beta-Thalassemia / Q000235:genetics* / Q000473:pathology / Q000628:therapy
D005319:Fetal Hemoglobin
colony assay; deletion; engineered nucleases; erythroid differentiation; fetal hemoglobin