提高细胞穿透锌指核酸酶蛋白用于精确基因组工程。
Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering.
机构信息
1] The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA [2] Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA [3] Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA [4] Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, China.
1] The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA [2] Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA [3] Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
出版信息
Mol Ther Nucleic Acids. 2015 Mar 10;4(3):e232. doi: 10.1038/mtna.2015.6.
Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4(+) T cells and 17% in CD34(+) hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20-30% in CD4(+) T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering.
为了使靶向基因组编辑充分发挥其在基础研究和医学领域的潜力，需要安全、高效和广泛适用的方法将特定部位的核酸酶递送到细胞内。我们之前曾报道过，锌指核酸酶（ZFN）蛋白具有透过细胞膜的固有能力，并可通过直接应用于人类细胞来诱导基因组修饰。在此，我们表明将串联核定位信号（NLS）重复序列整合到 ZFN 蛋白骨架中可使细胞通透性增强近 13 倍，并且单次给予多 NLS ZFN 蛋白可使 CD4(+)T 细胞中的基因组修饰率高达 26%，CD34(+)造血干/祖细胞中的基因组修饰率高达 17%。此外，我们表明多 NLS ZFN 蛋白可减弱脱靶效应，并且 ZFN 蛋白对的共递送可促进 CD4(+)T 细胞中双基因修饰频率达到 20-30%。这些结果说明了 ZFN 蛋白递送来进行精确基因组工程的适用性。
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