Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus.
Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus.
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DOI：
10.1093/nar/gku224
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发表时间：
2014-06
影响因子：
14.9
通讯作者：
Rawlings DJ
中科院分区：
文献类型：
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作者：
Wang Y;Khan IF;Boissel S;Jarjour J;Pangallo J;Thyme S;Baker D;Scharenberg AM;Rawlings DJ
LAGLIDADG 归巢核酸内切酶 (LHE) 是具有 20-22 bp 识别位点的紧凑型核酸内切酶，因此是用于基因组编辑应用的工程位点特异性 DNA 切割酶的理想支架。在这里，我们描述了一种 LHE 工程的通用方法，该方法将合理设计与定向进化相结合，使用酵母表面显示高通量切割选择。该方法用于改变 I-Anil LHE 的结合和切割特异性，以识别导致小鼠 X 连锁免疫缺陷 (XID)（人类 X 连锁无丙种球蛋白血症 (XLA) 模型）的小鼠布鲁顿酪氨酸激酶 (Btk) 基因突变。 I-AniI 所需的重新靶向涉及 DNA 接触界面的逐步重塑，以适应与天然切割序列的 9 个碱基差异。与野生型 (WT) 等位基因相比，渐进工程过程中出现的酶对 XID 突变等位基因具有特异性，并且在体外和细胞报告基因测定中表现出与 WT I-AniI 相当的活性。该酶与转录激活因子样效应器 (TALE) 的位点特异性 DNA 结合域融合，进一步提高了基因编辑效率。这些结果说明了 LHE 酶作为治疗性基因组工程的特异性和有效工具的潜力。
LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20–22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)—a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering.
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影响因子：
14.9
作者：
Jacoby, Kyle;Metzger, Michael;Scharenberg, Andrew M.
通讯作者：
Scharenberg, Andrew M.
影响因子：
14.9
作者：
Takeuchi, Ryo;Certo, Michael;Stoddard, Barry L.
通讯作者：
Stoddard, Barry L.
影响因子：
14.9
作者：
Jarjour J;West-Foyle H;Certo MT;Hubert CG;Doyle L;Getz MM;Stoddard BL;Scharenberg AM
通讯作者：
Scharenberg AM
影响因子：
14.9
作者：
Kuhar R;Gwiazda KS;Humbert O;Mandt T;Pangallo J;Brault M;Khan I;Maizels N;Rawlings DJ;Scharenberg AM;Certo MT
通讯作者：
Certo MT
影响因子：
64.8
作者：
通讯作者：
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