Genome editing in human hematopoietic stem and progenitor cells via CRISPR-Cas9-mediated homology-independent targeted integration.
Genome editing in human hematopoietic stem and progenitor cells via CRISPR-Cas9-mediated homology-independent targeted integration.
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DOI：
10.1016/j.ymthe.2020.12.010
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发表时间：
2021-04-07
期刊：
影响因子：
--
通讯作者：
Larochelle A
中科院分区：
文献类型：
--
作者：
Bloomer H;Smith RH;Hakami W;Larochelle A
造血干细胞和祖细胞(HSPC)的体外基因矫正已成为治疗遗传性人类血液疾病的一种有前途的治疗方法。使用工程核酸酶将治疗性转基因定位于其内源遗传位点，解决了与基于病毒载体的基因替换策略相关的许多限制，如插入突变、可变基因剂量和异位表达。核酸酶介导的位点特异性DNA整合的常见方法利用同源定向修复(HDR)途径。然而，这些方法在HSPC中效率低下，因为非同源末端连接(NHEJ)是主要的DNA修复机制。最近，一种基于NHEJ的新方法被称为同源非依赖靶向整合(HITI)，用于CRISPR-Cas9介导的转基因敲击，在非分裂细胞中显示出提高了位点特异性整合频率。在这里，我们利用一种基于HITI的方法，在动员的人外周血CD34+HSPC中实现强大的位点特异性转基因整合。作为概念的证明，使用重组腺相关病毒血清6型载体和单引导RNA/Cas9核糖核蛋白复合体将报告基因靶向于临床相关的遗传位点。我们将证明在移植到免疫缺陷小鼠后重新填充HSPC时，稳定的HITI介导的基因组编辑(∼21%)水平很高。我们的研究表明，HITI介导的基因组编辑为CD34+HSPC中基于HDR的转基因整合提供了一种有效的替代方案。基因组编辑的常见方法利用同源定向修复(HDR)途径。然而，这些方法在人类造血干细胞和祖细胞(HSPC)中是无效的，其中非同源末端连接(NHEJ)是主要的DNA修复机制。Blomer等人。使用一种被称为同源独立靶向整合(HITI)的基于∼的新方法，在人类重新填充HSPC中展示了稳定的高水平基因组编辑(NHEJ 21%)。
Ex vivo gene correction of hematopoietic stem and progenitor cells (HSPCs) has emerged as a promising therapeutic approach for treatment of inherited human blood disorders. Use of engineered nucleases to target therapeutic transgenes to their endogenous genetic loci addresses many of the limitations associated with viral vector-based gene replacement strategies, such as insertional mutagenesis, variable gene dosage, and ectopic expression. Common methods of nuclease-mediated site-specific integration utilize the homology-directed repair (HDR) pathway. However, these approaches are inefficient in HSPCs, where non-homologous end joining (NHEJ) is the primary DNA repair mechanism. Recently, a novel NHEJ-based approach to CRISPR-Cas9-mediated transgene knockin, known as homology-independent targeted integration (HITI), has demonstrated improved site-specific integration frequencies in non-dividing cells. Here we utilize a HITI-based approach to achieve robust site-specific transgene integration in human mobilized peripheral blood CD34+ HSPCs. As proof of concept, a reporter gene was targeted to a clinically relevant genetic locus using a recombinant adeno-associated virus serotype 6 vector and single guide RNA/Cas9 ribonucleoprotein complexes. We demonstrate high levels of stable HITI-mediated genome editing (∼21%) in repopulating HSPCs after transplantation into immunodeficient mice. Our study demonstrates that HITI-mediated genome editing provides an effective alternative to HDR-based transgene integration in CD34+ HSPCs. Common methods of genome editing utilize the homology-directed repair (HDR) pathway. However, these approaches are inefficient in human hematopoietic stem and progenitor cells (HSPCs), where non-homologous end joining (NHEJ) is the primary DNA repair mechanism. Bloomer et al. demonstrate stable high-level genome editing (∼21%) in human repopulating HSPCs using a novel NHEJ-based approach known as homology-independent targeted integration (HITI).
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影响因子：
8.8
作者：
Kuo CY;Long JD;Campo-Fernandez B;de Oliveira S;Cooper AR;Romero Z;Hoban MD;Joglekar AV;Lill GR;Kaufman ML;Fitz-Gibbon S;Wang X;Hollis RP;Kohn DB
通讯作者：
Kohn DB
影响因子：
64.8
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Naldini, Luigi
影响因子：
14.8
作者：
Bak RO;Dever DP;Porteus MH
通讯作者：
Porteus MH
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
20.3
作者：
Kang, Elizabeth M.;Choi, Uimook;Malech, Harry L.
通讯作者：
Malech, Harry L.