Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.
Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.
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DOI：
10.1038/nbt.3408
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发表时间：
2015-12
影响因子：
46.9
通讯作者：
Cannon PM
中科院分区：
文献类型：
--
作者：
Wang J;Exline CM;DeClercq JJ;Llewellyn GN;Hayward SB;Li PW;Shivak DA;Surosky RT;Gregory PD;Holmes MC;Cannon PM
使用与断裂位点同源的靶向核酸酶和DNA供体模板进行基因组编辑已被证明在人类造血干细胞和祖细胞（HSPC）中具有挑战性，特别是在最原始的长期再增殖细胞群中。在这里，我们报告了锌指核酸酶（ZFN）mRNA的电穿孔与通过AAV血清型6载体的供体模板递送的组合指导HSPC中的有效基因组编辑，在动员的外周血CD34 + HSPC中以17%和26%的平均频率分别在CCR 5和AAVS1基因座处实现GFP盒的位点特异性插入，并且在胎肝HSPC中以19%和43%的平均频率插入。分别值得注意的是，这种方法修饰了CD34 + CD133 + CD90+细胞群，这是含有长期再增殖造血干细胞（HSC）的CD34+细胞的次要组分。基因组编辑的HSPC也长期移植到免疫缺陷小鼠中，证实了这种方法靶向HSC。我们的研究结果为基因组编辑技术在HSPC中更强大的应用提供了策略。
Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by AAV serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome editing technologies in HSPCs.