Inducible multiplexed CRISPR interference system in human embryonic stem cells
10.3969/j.issn.1674-8115.2019.05.007
- Author:
Chao-Nan ZHU
1
Author Information
1. Department of Histoembryology, Genetics & Development, Shanghai Jiao Tong University College of Basic Medical Sciences
- Publication Type:Journal Article
- Keywords:
CRISPR interference system;
Golden Gate assembly;
Human embryonic stem cells (hESCs);
Inducible multiple gene knockdown
- From:
Journal of Shanghai Jiaotong University(Medical Science)
2019;39(5):478-486
- CountryChina
- Language:Chinese
-
Abstract:
Objective • To generate a doxycycline (Dox)-inducible multiplexed CRISPR interference (CRISPRi) system for multiple gene inhibition in human embryonic stem cells (hESCs) to explore the function of gene families and model multigene diseases. Methods • A Dox-inducible multiplexed CRISPRi system was developed by Golden Gate assembly in hESCs. This system consisted of two plasmids, one expressing modified repressive nuclease-deactivated CRISPR-associated protein 9 (dCas9) and Krüppel-associated box (KRAB) transcriptional repressor domain under the control of Dox, the other carrying eight independent guide RNA (gRNA) expression cassettes. PCR was conducted using total genomic DNA as a template to confirm whether these two plasmids were integrated into genome. Western blotting was performed to confirm whether the expression of dCas9-KRAB could be induced by Dox treatment. Results • Using this tunable CRISPRi system, multiple genes were successfully silenced simultaneously in hESCs. The silence of genes and related to hESC self-renewal caused obvious cell differentiation in terms of changed cell morphology, decreased activity of alkaline phosphatase, and reduced expression of stage-specific embryonic antigen 4 (SSEA4), a marker of undifferentiated hESCs. Conclusion • This Dox-inducible multiplexed CRISPRi system can be used for quick and efficient silence of multiple genes in hESCs in a highly controlled manner.
