Ye Lin, Wang Jiaming, Tan Yuting, Beyer Ashley I, Xie Fei, Muench Marcus O, Kan Yuet Wai
Department of Medicine, University of California, San Francisco, CA 94143; Institute for Human Genetics, University of California, San Francisco, CA 94143;
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;
Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10661-5. doi: 10.1073/pnas.1612075113. Epub 2016 Sep 6.
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.
胎儿血红蛋白遗传性持续存在（HPFH）是一些个体终生都有高水平胎儿血红蛋白的一种状况。患有复合杂合β地中海贫血或镰状细胞病（SCD）以及HPFH的个体临床表现较轻。利用RNA引导的成簇规律间隔短回文重复序列相关Cas9（CRISPR-Cas9）基因组编辑技术，我们在正常造血干细胞和祖细胞（HSPCs）中删除了13 kb的β珠蛋白基因座，以模拟自然发生的西西里HPFH突变。在上游和下游断点引导RNA（gRNA）引导的金黄色葡萄球菌Cas9核酸酶（SaCas9）均递送的细胞中，靶向删除效率达到31%。与未删除的集落相比，由具有HPFH删除的HSPCs分化而来的红系集落显示出显著更高的γ珠蛋白基因表达。通过T7核酸内切酶1检测，我们在删除的集落中未检测到任何脱靶效应。我们提出，这种使用非同源末端连接（NHEJ）来修饰基因组的策略可能为开发针对纯合β地中海贫血和SCD患者的安全自体移植提供一种有效方法。
