A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells.
A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells.
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多能干细胞（PSC）为探索人类生物学提供了令人兴奋的资源；然而，在包括干细胞在内的许多细胞类型中，基因编辑效率仍然相对较低。使用 CRISPR-Cas9 系统进行基因编辑提供了一种有吸引力的解决方案，该解决方案改进了以前的基因编辑方法；然而，与其他技术一样，脱靶诱变仍然是一个令人担忧的问题。高保真 Cas9 变体极大地减少了脱靶诱变，并为该问题提供了解决方案。为了评估它们作为基于细胞的基因编辑平台的一部分的实用性，使用集成到 AAVS1 安全港基因座的高保真 (HF) 四环素诱导工程化脓性链球菌 SpCas9 (HF-iCas9) 生成了人类 PSC 系。通过对细胞进行工程改造，使其具有 Cas9 的可控表达，我们无需在细胞转染过程中加入大型 Cas9 表达质粒。通过将 DNA 靶向引导 RNA (gRNA) 和供体片段包装到单个质粒主链中，进一步优化了遗传货物的递送。证明了基于同源定向修复 (HDR) 的 CLYBL 安全港位点基因敲入以及内源 SOX2 和 SIX6 基因的潜力。此外，我们使用非同源末端连接（NHEJ）来敲除疾病相关等位基因。这些高保真 CRISPR 工具和由此产生的 HF-iCas9 细胞系将有助于跨不同遗传背景的细胞类型报告基因和突变体的生产。
Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.
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一个 iCRISPR 平台，用于在人类多能干细胞中进行快速、可多重、可诱导的基因组编辑。
影响因子：
23.9
作者：
Gonzalez, Federico;Zhu, Zengrong;Shi, Zhong-Dong;Lelli, Katherine;Verma, Nipun;Li, Qing V.;Huangfu, Danwei
通讯作者：
Huangfu, Danwei
影响因子：
12.3
作者：
Dang Y;Jia G;Choi J;Ma H;Anaya E;Ye C;Shankar P;Wu H
通讯作者：
Wu H
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
2.8
作者：
Pourcel, C;Salvignol, G;Vergnaud, G
通讯作者：
Vergnaud, G
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle