Comparison of Zinc Finger Nucleases Versus CRISPR-Specific Nucleases for Genome Editing of the Wiskott-Aldrich Syndrome Locus
Comparison of Zinc Finger Nucleases Versus CRISPR-Specific Nucleases for Genome Editing of the Wiskott-Aldrich Syndrome Locus
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DOI：
10.1089/hum.2017.047
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发表时间：
2017-10-25
影响因子：
4.2
通讯作者：
Martin, Francisco
中科院分区：
文献类型：
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作者：
Gutierrez-Guerrero, Alejandra;Sanchez-Hernandez, Sabina;Martin, Francisco
包括Wiskott-Aldrich综合征(WAS)在内的原发免疫缺陷是使用特异性核酸酶(SNS)进行基因组编辑策略的主要目标，因为少量校正的造血干细胞可以治愈患者。在这项工作中，我们设计了不同的IS基因特异的CRISPR/Cas9系统，并以K-562细胞为细胞模型，以质粒核重组或整合缺陷慢病毒载体(IDLV)为载体，比较了它们与同源二聚体和异源二聚体的锌指核酸酶(ZFN)的效率和特异性。不同的CRISPR/Cas9和ZFN SNS在使用质粒核转染法时表现出相似的效率。然而，表达ZFN的双重IDLV比表达Cas9和引导RNA的双重IDLV或在同一载体中表达Cas9和引导RNA的一体化IDLV更有效。异二聚体ZFN和CRISPR/Cas9的特异性，通过在AS编辑的细胞中伽马-H_2AX焦点形成的增量来衡量，对于两者来说是相似的，并且两者的表现都优于同源二聚体ZFN，独立于所使用的递送系统。有趣的是，我们发现使用IDLV传递SNS比用质粒核转染法更有效，遗传毒性更小。我们还展示了异源二聚体ZFN和CRISPR/Cas9在同源导向的基因敲入策略中的相似行为，分别有88%和83%的供体插入到IS基因座，而当使用同源二聚体ZFNS时，只有45%的插入在靶点上。综上所述，我们的数据表明CRISPR/Cas9和异二聚体ZFN都是进一步发展基于SN的AS基因治疗策略的良好替代方案。然而，在这项研究中，IDLV递送IS特异性异二聚体ZFN是所有系统中最好的选择。
Primary immunodeficiencies, including Wiskott-Aldrich syndrome (WAS), are a main target for genome-editing strategies using specific nucleases (SNs) because a small number of corrected hematopoietic stem cells could cure patients. In this work, we have designed various WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific zinc finger nucleases (ZFNs), using K-562 cells as a cellular model and plasmid nucleofection or integration-deficient lentiviral vectors (IDLVs) for delivery. The various CRISPR/Cas9 and ZFN SNs showed similar efficiency when using plasmid nucleofection for delivery. However, dual IDLVs expressing ZFNs were more efficient than dual IDLVs expressing Cas9 and guide RNA or all-in-one IDLVs, expressing Cas9 and guide RNA in the same vector. The specificity of heterodimeric ZFNs and CRISPR/Cas9, measured by increments in gamma-H2AX focus formation in WAS-edited cells, was similar for both, and both outperformed homodimeric ZFNs independently of the delivery system used. Interestingly, we show that delivery of SNs, using IDLVs, is more efficient and less genotoxic than plasmid nucleofection. We also show the similar behavior of heterodimeric ZFNs and CRISPR/Cas9 for homology-directed gene knock-in strategies, with 88 and 83% of the donors inserted in the WAS locus, respectively, whereas when using homodimeric ZFNs only 45% of the insertions were on target. In summary, our data indicate that CRISPR/Cas9 and heterodimeric ZFNs are both good alternatives to further develop SN-based gene therapy strategies for WAS. However, IDLV delivery of WAS-specific heterodimeric ZFNs was the best option of all systems compared in this study.