Liu Jia, Gaj Thomas, Patterson James T, Sirk Shannon J, Barbas Carlos F
The Departments of Chemistry and Cell and Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America.
PLoS One. 2014 Jan 20;9(1):e85755. doi: 10.1371/journal.pone.0085755. eCollection 2014.
Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.
转录激活样效应物核酸酶（TALENs）能够将靶向基因改变引入多种细胞系和生物体中。这些可定制的核酸酶由源自TAL效应蛋白的可编程序列特异性DNA结合模块与非特异性FokI切割结构域融合而成。将这些核酸酶导入细胞已被证明具有挑战性，因为TAL效应物DNA结合结构域的大尺寸和高度重复性使其无法整合到许多类型的病毒载体中。此外，病毒和非病毒基因递送方法存在插入诱变的风险，并且已被证明会增加位点特异性核酸酶的脱靶活性。我们之前证明，直接递送锌指核酸酶蛋白能够在多种哺乳动物细胞类型中实现高效基因敲除，且脱靶效应降低。在此我们表明，将细胞穿透性聚精氨酸肽与每个TAL效应物重复序列上存在的表面暴露半胱氨酸残基偶联，赋予了纯化的TALEN蛋白细胞穿透活性。这些修饰在还原条件下是可逆的，并且能够以与TALEN表达载体瞬时转染相当的效率实现TALEN介导的人类CCR5和BMPR1A基因敲除。这些发现表明，通过将化学官能团偶联到TALEN蛋白表面来促进直接蛋白质递送，是目前将TALEN递送至哺乳动物细胞的非病毒和病毒方法的一种有前景的替代方法。
