Sui Ming, Zhou Meiling, Cui Mengge, Liu Huan, Zhang Xiaolin, Hu Na, Li Yang, Wang Beibei, Yang Guojun, Gui Pengling, Zhu Lingqiang, Wan Feng, Zhang Bin
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
The Institute for Brain Research, Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan, 430030, China.
Cell Mol Life Sci. 2025 Jun 23;82(1):249. doi: 10.1007/s00018-025-05786-7.
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 The inducible transcriptional manipulation could be restored to its original level when 4OHT was withdrawn.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.
CRISPR激活与干扰（CRISPRa/i）是通过将无核酸酶活性的Cas9（dCas9）与由小向导RNA（sgRNA）引导的转录调节因子融合，从而在哺乳动物细胞和小鼠中调节转录的高效工具。然而，仍需要一种可控的基因调控系统来研究和操纵动态生物学过程。在此，我们报道了一种灵活的药物响应型CRISPRa/i系统，该系统通过将突变的人雌激素受体（ERT2）结构域与CRISPRa/i组件融合，实现转录调控，ERT2结构域对雌激素类似物他莫昔芬或其活性代谢物4-羟基他莫昔芬（4OHT）有响应。用4OHT处理后，最佳变体ERT2-ERT2-CRISPRa/i-ERT2（iCRISPRa/i）显示iCRISPRa/i从细胞质快速转运至细胞核，并随后产生转录反应。当撤去4OHT时，可诱导的转录操纵可恢复到其原始水平。此外，iCRISPRa/i的基因表达调控效率与非诱导型和强力霉素诱导型相当，且渗漏率更低，药物反应活性更快。iCRISPRa/i系统成功地在多种细胞系中诱导了表型变化。这些结果表明，iCRISPRa/i系统可以实现快速且灵活的药物响应型转录调控和表型变化，从而为功能获得和功能缺失模型构建以及基因治疗提供更好的选择。
