反式激活crRNA
Cas9
核糖核蛋白
三元络合物
引导RNA
转染
清脆的
基因组编辑
生物
细胞生物学
化学
分子生物学
核糖核酸
基因
生物化学
酶
作者
Seung Min Kim,Sang Chul Shin,Eunice EunKyeong Kim,Sang‐Heon Kim,Kwideok Park,Se H. Oh,Mihue Jang
出处
期刊:ACS Nano
[American Chemical Society]
日期:2018-07-20
卷期号:12 (8): 7750-7760
被引量:46
标识
DOI:10.1021/acsnano.8b01670
摘要
Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for in vivo applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing in vivo. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both a nuclear localization sequence and a low-molecular-weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNP) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) via in vitro and in vivo editing of the KRAS gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and high-throughput genetic screening.
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