Novel drug-inducible CRISPRa/i systems for rapid and reversible manipulation of gene transcription

Novel drug-inducible CRISPRa/i systems for rapid and reversible manipulation of gene transcription

Abstract CRISPR activation and interference (CRISPRa/i) are highly effective tools to regulate transcription by fusing dead Cas9 (dCas9) with transcriptional regulatory factors guided by small guide RNA (sgRNA) in mammalian cells and mice. Still, a controllable gene regulation system is desired to i...

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Bibliographic Details
Main Authors: Ming Sui, Meiling Zhou, Mengge Cui, Huan Liu, Xiaolin Zhang, Na Hu, Yang Li, Beibei Wang, Guojun Yang, Pengling Gui, Lingqiang Zhu, Feng Wan, Bin Zhang
Format: Article
Language:English
Published: Springer 2025-06-01
Series:Cellular and Molecular Life Sciences
Subjects:
iCRISPRa/i
4OHT
Transcriptional regulation
Phenotypic change
Online Access:https://doi.org/10.1007/s00018-025-05786-7
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Summary:Abstract CRISPR activation and interference (CRISPRa/i) are highly effective tools to regulate transcription by fusing dead Cas9 (dCas9) with transcriptional regulatory factors guided by small guide RNA (sgRNA) in mammalian cells and mice. Still, a controllable gene regulation system is desired to investigate and manipulate dynamic biological processes. Here, we reported flexible drug-responsive CRISPRa/i systems by fusing mutated human estrogen receptor (ERT2) domains, which responded to estrogen analogue tamoxifen or its active metabolite 4-hydroxy-tamoxifen (4OHT), to CRISPRa/i components for transcriptional regulation. Upon 4OHT treatment, the optimal variants, ERT2-ERT2-CRISPRa/i-ERT2 (iCRISPRa/i), showed rapid protein translocation of iCRISPRa/i from cytoplasm to nucleus and subsequent transcriptional response. The inducible transcriptional manipulation could be restored to its original level when 4OHT was withdrawn. Moreover, the efficiencies of gene expression regulation of iCRISPRa/i were comparable to those of non-inducible and doxycycline-inducible counterparts, with a lower leakage and a faster drug response activity. The iCRISPRa/i systems successfully induced phenotypic changes in various cell lines. These results highlight that iCRISPRa/i systems could achieve fast and flexible drug-responsive transcriptional modulation and phenotypic changes, and thus provide better options for gain- and loss-of-function model construction and gene therapy.
ISSN:1420-9071

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