Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs.
Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs.
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DOI：
10.1242/dev.138081
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发表时间：
2016-12-01
期刊：
影响因子：
--
通讯作者：
Vallier L
中科院分区：
文献类型：
--
作者：
Bertero A;Pawlowski M;Ortmann D;Snijders K;Yiangou L;Cardoso de Brito M;Brown S;Bernard WG;Cooper JD;Giacomelli E;Gambardella L;Hannan NR;Iyer D;Sampaziotis F;Serrano F;Zonneveld MC;Sinha S;Kotter M;Vallier L
诱导基因功能丧失实验对于揭示发育、生理和疾病的潜在机制是必要的。然而，目前的方法是复杂的，缺乏鲁棒性，并不适用于多种细胞类型。在这里，我们通过开发单步优化的诱导型基因敲低或敲除（sOPTiKD或sOPTiKO）平台来解决这些限制。这些都是基于人类基因组安全港的基因工程，结合改进的四环素诱导系统和CRISPR/Cas9技术。我们证实了这些方法在人多能干细胞（hPSC）中的功效，并表明sOPTiKD/KO hPSC的产生简单、快速，并且允许hPSC和多种分化细胞中严格控制的单个或多重基因敲低或敲除。最后，我们通过研究转录因子（OCT 4和T），细胞周期调节因子（细胞周期蛋白D家族成员）和表观遗传修饰剂（DPY 30）的功能来说明这种方法的普遍适用性。总体而言，sOPTiKD和sOPTiKO为与人类发育研究相关的多种细胞类型的功能分析提供了独特的机会。 开发了新型优化的诱导型敲低和敲除平台，并用于评估人多能干细胞及其分化后代中的基因功能。
Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development. Novel optimized inducible knockdown and knockout platforms are developed and used to assess gene function in human pluripotent stem cells and their differentiated progeny.
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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：
23.9
作者：
Mandal, Pankaj K.;Ferreira, Leonardo M. R.;Collins, Ryan;Meissner, Torsten B.;Boutwell, Christian L.;Friesen, Max;Vrbanac, Vladimir;Garrison, Brian S.;Stortchevoi, Alexei;Bryder, David;Musunuru, Kiran;Brand, Harrison;Tager, Andrew M.;Allen, Todd M.;Talkowski, Michael E.;Rossi, Derrick J.;Cowan, Chad A.
通讯作者：
Cowan, Chad A.
影响因子：
3.7
作者：
Fath S;Bauer AP;Liss M;Spriestersbach A;Maertens B;Hahn P;Ludwig C;Schäfer F;Graf M;Wagner R
通讯作者：
Wagner R
影响因子：
46.9
作者：
Hockemeyer, Dirk;Soldner, Frank;Beard, Caroline;Gao, Qing;Mitalipova, Maisam;DeKelver, Russell C.;Katibah, George E.;Amora, Ranier;Boydston, Elizabeth A.;Zeitler, Bryan;Meng, Xiangdong;Miller, Jeffrey C.;Zhang, Lei;Rebar, Edward J.;Gregory, Philip D.;Urnov, Fyodor D.;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
影响因子：
12.4
作者：
Herbst, Friederike;Ball, Claudia R.;Glimm, Hanno
通讯作者：
Glimm, Hanno