Liu Jia, Liang Ya-Jun, Ren Pei-Ling, Gaj Thomas
Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, China.
Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai, China.
Methods Mol Biol. 2018;1867:253-273. doi: 10.1007/978-1-4939-8799-3_19.
Genome-editing technologies have revolutionized the biomedical sciences by providing researchers with the ability to quickly and efficiently modify genes. While programmable nucleases can be introduced into cells using a variety of techniques, their delivery as purified proteins is an effective approach for limiting off-target effects. Here, we describe step-by-step procedures for manufacturing and delivering genome-modifying proteins-including Cas9 ribonucleoproteins (RNPs) and TALE and zinc-finger nucleases-into mammalian cells. Protocols for combining Cas9 RNP with naturally recombinogenic adeno-associated virus (AAV) donor vectors for the seamless insertion of transgenes by homology-directed genome editing are also provided.
基因组编辑技术为研究人员提供了快速高效地修饰基因的能力，从而彻底改变了生物医学科学。虽然可编程核酸酶可以通过多种技术导入细胞，但将其作为纯化蛋白递送是限制脱靶效应的有效方法。在这里，我们描述了将基因组修饰蛋白（包括Cas9核糖核蛋白（RNP）、转录激活因子样效应物核酸酶（TALE）和锌指核酸酶）制造并递送至哺乳动物细胞的分步程序。还提供了将Cas9 RNP与天然重组腺相关病毒（AAV）供体载体相结合的方案，用于通过同源定向基因组编辑无缝插入转基因。
