Marker-free coselection for CRISPR-driven genome editing in human cells
Marker-free coselection for CRISPR-driven genome editing in human cells
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DOI：
10.1038/nmeth.4265
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发表时间：
2017-06-01
期刊：
影响因子：
48
通讯作者：
Doyon, Yannick
中科院分区：
文献类型：
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作者：
Agudelo, Daniel;Duringer, Alexis;Doyon, Yannick
靶向基因组编辑能够为生物研究创建真正的细胞模型，并可应用于基于人类细胞的疗法。因此，非常需要广泛适用和通用的方法来提高其在细胞群中的功效。我们设计了一种简单而强大的共选择策略，通过利用 CRISPR Cas9 和 Cpfl 系统的多重功能，通过核酸酶驱动的非同源末端连接 (NH EJ) 或同源定向修复 (HDR) 事件来富集细胞。选择使细胞对哇巴因具有抗性的普遍存在的钠/钾泵（Na+/K+ ATP酶）的显性等位基因，用于富集另一个未连锁的感兴趣位点的定制遗传修饰，从而有效地提高工程细胞的回收率。该过程很容易适用于转化细胞和原代细胞，包括造血干细胞和祖细胞。通用 CRISPR 试剂和市售小分子抑制剂的使用简化了人类细胞中无标记遗传变化的整合。
Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (N H EJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR Cas9 and Cpfl systems. Selection for dominant alleles of the ubiquitous sodium/ potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.