结合CRISPR激活和干扰功能使用dCas9和G-四重复结构
Mohammad Lutful Kabir1, Sineth G Kodikara2, Mohammed Enamul Hoque1
1Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, United States.
NAR molecular medicine
|February 5, 2025
概括
集群定期间隔的短平行体重复 (CRISPR) 干扰和激活,针对dCas9有效调节基因表达的c-Myc促进体. 这种CRISPR-dCas9系统在细胞和体外研究中显示出显著的c-Myc抑制和激活.
科学领域:
- 分子生物学分子生物学
- 基因规则 基因规则
- 生物物理学的生物物理.
背景情况:
- c-Myc瘤基因是细胞增殖的关键调节者,其失调与各种癌症有关,包括伯基特淋巴瘤.
- 促进体区域中的G-四重复 (GQ) 结构可以影响基因转录.
- 克里斯普尔干扰 (克里斯普尔i) 和克里斯普尔激活 (克里斯普尔a) 是基因调节的强大工具.
研究的目的:
- 为了研究利用核酶死Cas9 (dCas9) 调节基因的c-Myc促进体中准G-四重复形成序列 (PQS) 的疗效.
- 为了实现CRISPR介导的转录抑制和c-Myc在RNA和蛋白质水平上的激活.
- 阐明CRISPR-dCas9与c-Myc促进体相互作用的机制细节及其对转录的影响.
主要方法:
- 使用dCas9的CRISPR干扰 (CRISPRi) 和CRISPR激活 (CRISPRa) 系统来准c-Myc促进器中的一个PQS.
- 在伯基特淋巴瘤细胞系和体外实验中进行实验.
- 采用定量实时PCR (qRT-PCR) 进行mRNA分析,用于蛋白质水平评估的西部涂抹和细胞活力测试.
- 进行了广泛的体外生物物理研究,以分析分子相互作用.
主要成果:
- 用dCas9针对PQS附近的模板链破坏了GQ的稳定,导致c-Myc mRNA (2.1倍) 和蛋白质 (1.6倍) 水平显著增加.
- 针对非模板链 (NTS) 上的个别位置,dCas9降低了c-Myc mRNA (1.8倍) 和蛋白质 (2.5倍) 的水平.
- 同时针对两个NTS位点导致了实质性的抑制:mRNA的3.6倍和蛋白质的9.8倍.
- 细胞活力测定显示,单个和双重NTS向的细胞活力测定显示相应的减少 (分别是1.7倍和4.7倍).
- 试管生物物理研究量化支持了细胞发现,并提供了机理性的见解.
结论:
- CRISPR-dCas9对c-Myc促进体的向,特别是在PQS附近,是激活和抑制基因的有效策略.
- 通过dCas9调节GQ稳定性在调节c-Myc转录中起着至关重要的作用.
- 这种方法为控制瘤基因表达提供了一种强大而通用的方法,具有潜在的治疗意义.
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