Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia
Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia
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DOI：
10.1073/pnas.1612075113
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发表时间：
2016-09-20
影响因子：
11.1
通讯作者：
Kan, Yuet Wai
中科院分区：
文献类型：
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作者：
Ye, Lin;Wang, Jiaming;Kan, Yuet Wai
胎儿血红蛋白的遗传持续性（HPFH）是在一些个体中终生具有高水平的胎儿血红蛋白的一种状况。患有复合杂合子β-地中海贫血或镰状细胞病（SCD）和HPFH的个体具有较轻的临床表现。使用RNA引导的成簇规则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（HSPC）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。通过递送上游和下游断裂点引导RNA（gRNA）引导的金黄色葡萄球菌Cas9核酸酶（SaCas 9），在细胞中靶向缺失的效率达到31%。从HPFH缺失的HSPCs分化的红系细胞集落显示与未缺失的集落相比显著更高的γ-珠蛋白基因表达。通过T7核酸内切酶1测定，我们在缺失的菌落中未检测到任何脱靶效应。我们认为，这种使用非同源末端连接（NHEJ）来修饰基因组的策略可能为纯合子β-地中海贫血和SCD患者提供一种安全的自体移植的有效方法。
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 beta-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 beta-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 gamma-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 beta-thalassemia and SCD.