Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs

被引：55

作者：

Bertero, Alessandro ^{[1
,2
,7
]}

Pawlowski, Matthias ^{[1
,3
]}

Ortmann, Daniel ^{[1
,2
]}

Snijders, Kirsten ^{[1
,2
]}

Yiangou, Loukia ^{[1
,4
]}

de Brito, Miguel Cardoso ^{[1
,2
]}

Brown, Stephanie ^{[1
,2
]}

Bernard, William G. ^{[1
,4
]}

Cooper, James D. ^{[1
,4
]}

Giacomelli, Elisa ^{[1
,2
]}

Gambardella, Laure ^{[1
,4
]}

Hannan, Nicholas R. F. ^{[1
,2
,6
]}

Iyer, Dharini ^{[1
,4
]}

Sampaziotis, Fotios ^{[1
,2
]}

Serrano, Felipe ^{[1
,4
]}

Zonneveld, Marielle C. F. ^{[1
,2
]}

Sinha, Sanjay ^{[1
,4
]}

Kotter, Mark ^{[1
,3
]}

Vallier, Ludovic ^{[1
,2
,5
]}

机构：

[1] Univ Cambridge, Wellcome Trust MRC Stem Cell Inst, Anne McLaren Lab, Cambridge CB2 0SZ, England

[2] Univ Cambridge, Dept Surg, Cambridge CB2 0QQ, England

[3] Univ Cambridge, Dept Clin Neurosci, Cambridge CB2 0QQ, England

[4] Univ Cambridge, Div Cardiovasc Med, Cambridge, England

[5] Wellcome Trust Sanger Inst, Hinxton CB10 1SA, England

[6] Univ Nottingham, Ctr Biomol Sci, Nottingham NG7 2RD, England

[7] Univ Washington, Dept Pathol, Seattle, WA 98109 USA

来源：

DEVELOPMENT | 2016年 / 143卷 / 23期

基金：

英国惠康基金; 英国医学研究理事会; 欧洲研究理事会;

关键词：

Human pluripotent stem cells; shRNA; CRISPR/Cas9; OCT4; POU5F1; T; brachyury; DPY30; EMBRYONIC STEM-CELLS; MEDIATED CASSETTE EXCHANGE; AAVS1; LOCUS; GENE; DIFFERENTIATION; MOUSE; BRACHYURY; SPECIFICATION; INTERFERENCE; GENERATION;

D O I：

10.1242/dev.138081

中图分类号：

Q [生物科学];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页码：4405 / 4418

页数：14

共 55 条

[1] Pluripotent stem cells in disease modelling and drug discovery [J].