›› 2019, Vol. 39 ›› Issue (5): 478-.doi: 10.3969/j.issn.1674-8115.2019.05.007

• Original article (Basic research) • Previous Articles     Next Articles

Inducible multiplexed CRISPR interference system in human embryonic stem cells

ZHU Chao-nan, CHEN Qin-wen, XIN Chen-ge, LI Hui   

  1. Department of Histoembryology, Genetics & Development, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
  • Online:2019-05-28 Published:2019-07-26
  • Supported by:
    Natural Science Foundation of Shanghai, 19ZR1428300

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 developedGolden Gate assembly in hESCs. This system consisted of two plasmids, one expressing modified repressive nucleasedeactivated 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) 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 of dCas9-KRAB could be inducedDox 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 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.

Key words: CRISPR interference system, inducible multiple gene knockdown, Golden Gate assembly, human embryonic stem cells (hESCs)

CLC Number: