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相关概念视频

CRISPR01:59

CRISPR

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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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

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概括
此摘要是机器生成的。

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.

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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.