Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.
Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.
复制标题
DOI：
10.1101/gr.171264.113
复制
发表时间：
2014-06
期刊：
影响因子：
7
通讯作者：
Kim H
中科院分区：
文献类型：
--
作者：
Ramakrishna S;Kwaku Dad AB;Beloor J;Gopalappa R;Lee SK;Kim H
来自CRISPR/Cas系统的rna引导内切酶（RGENs）是基因组编辑的有效工具。RGENs由Cas9蛋白和引导RNA两部分组成。质粒介导的将这些成分传递到细胞中可能导致质粒序列不受控制地整合到宿主基因组中，以及由细菌序列引起的不必要的免疫反应和潜在的安全问题。此外，这种递送方法需要转染工具。本研究表明，用细胞穿透肽（CPP）偶联的重组Cas9蛋白和CPP复合物引导rna进行简单处理可导致人类细胞系的内源性基因破坏。Cas9蛋白通过硫醚键与CPP结合，而引导RNA则与CPP络合，形成凝聚的带正电的纳米颗粒。用修饰的Cas9和引导RNA同时和顺序处理人类细胞，包括胚胎干细胞、真皮成纤维细胞、HEK293T细胞、HeLa细胞和胚胎癌细胞，导致有效的基因破坏，相对于质粒转染减少脱靶突变，从而产生含有rgen诱导突变的克隆。我们的cpp介导的RGEN传递过程提供了一种无质粒和额外的转染试剂的方法，以减少脱靶效应。我们设想我们的方法将促进rgen导向的基因组编辑。
RNA-guided endonucleases (RGENs) derived from the CRISPR/Cas system represent an efficient tool for genome editing. RGENs consist of two components: Cas9 protein and guide RNA. Plasmid-mediated delivery of these components into cells can result in uncontrolled integration of the plasmid sequence into the host genome, and unwanted immune responses and potential safety problems that can be caused by the bacterial sequences. Furthermore, this delivery method requires transfection tools. Here we show that simple treatment with cell-penetrating peptide (CPP)–conjugated recombinant Cas9 protein and CPP-complexed guide RNAs leads to endogenous gene disruptions in human cell lines. The Cas9 protein was conjugated to CPP via a thioether bond, whereas the guide RNA was complexed with CPP, forming condensed, positively charged nanoparticles. Simultaneous and sequential treatment of human cells, including embryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinoma cells, with the modified Cas9 and guide RNA, leads to efficient gene disruptions with reduced off-target mutations relative to plasmid transfections, resulting in the generation of clones containing RGEN-induced mutations. Our CPP-mediated RGEN delivery process provides a plasmid-free and additional transfection reagent–free method to use this tool with reduced off-target effects. We envision that our method will facilitate RGEN-directed genome editing.
登录
查看更多内容
影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1073/pnas.1003919107
发表时间：
2010-10-19
影响因子：
11.1
作者：
Ding, Hui;Inoue, Satoshi;Ljubimova, Julia Y.
通讯作者：
Ljubimova, Julia Y.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo