结合CRISPR激活和干扰能力使用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, USA.
bioRxiv : the preprint server for biology
|November 28, 2024
概括
针对dCas9有效调节基因表达的c-Myc促进体中的G-四重复序列的CRISPR干扰和激活. 这种方法显示了控制c-Myc水平和细胞活力的潜力.
科学领域:
- 分子生物学分子生物学
- 基因规则 基因规则
- 生物技术是生物技术.
背景情况:
- 该c-Myc瘤基因在细胞增殖中起着至关重要的作用,在癌症中经常出现失调.
- 发起地区的G-四重复形成序列 (PQS) 正成为关键的监管元素.
- 基于CRISPR的工具提供了精确的基因组编辑功能.
研究的目的:
- 为了研究利用核酶死Cas9 (dCas9) 对基因调节的c-Myc促进体向PQS的有效性.
- 为了证明CRISPR干扰 (CRISPRi) 和CRISPR激活 (CRISPRa) 在c-Myc位置.
- 阐明dCas9与PQS相互作用的机制细节及其对转录的影响.
主要方法:
- 利用dCas9来准伯基特淋巴瘤细胞系中的c-Myc促进体中PQS附近的区域.
- 通过针对非模板链来使用CRISPR干扰,通过针对模板链来激活CRISPR.
- 进行了体外生物物理研究,以补充细胞测定和理解转录调制.
主要成果:
- 用dCas9准模板链破坏了G-四重复的稳定性,使c-Myc mRNA和蛋白分别增加了2.1倍和1.6倍.
- 用dCas9针对非模板链减少了c-Myc mRNA和蛋白质的1.8倍和2.5倍,双位点向实现了3.6倍和9.8倍的减少.
- 细胞活力测试显示,非模板链向的细胞活力测试显示了相应的减少,表明了功能后果.
结论:
- 通过CRISPR-dCas9对c-Myc促进体PQS的向,可以实现强大的基因激活和抑制.
- 这项研究提供了一种新的策略,通过G-quadruplex操纵调节瘤基因表达.
- 这些发现得到了体外生物物理数据的支持,为转录控制提供了机理性的见解.
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