B组链球菌Cas9变种提供了可编程基因抑制和CRISPR-Cas转录效应的洞察力
Kathyayini P Gopalakrishna1, Gideon H Hillebrand2, Venkata H Bhavana1
1University of Pittsburgh School of Medicine, Department of Pediatrics, Pittsburgh, PA, USA.
Communications biology
|June 9, 2023
概括
B组链球菌Cas9通过非特异性DNA结合影响细菌的转录,而不是它的切割功能. 这一发现有助于开发新的工具来研究B组链球菌 (GBS) 基因功能.
科学领域:
- 微生物学 微生物学
- 遗传学 遗传学 是一个
- 分子生物学分子生物学
背景情况:
- B组链球菌 (GBS) 在新生儿和成年人中引起严重感染.
- GBS有一个CRISPR-Cas9系统,通常用于外来DNA防御.
- 最近的研究表明,GBS Cas9影响的基因转录超出了其内核酶活性.
研究的目的:
- 调查GBS Cas9影响全基因组转录的机制.
- 为了确定DNA结合或催化活动是否驱动这些转录效应.
- 开发一种用于GBS的向基因调节的工具.
主要方法:
- 生成的同源性GBS变体:Δcas9 (删除),dCas9 (催化不活跃) 和scas9 (DNA结合受损).
- 进行全基因组RNA测序 (RNA-seq) 来比较转录特征.
- 使用基于等离子体的单导向RNA系统与dCas9进行基因抑制.
主要成果:
- 通过Cas9结合非特异性原体空间邻基因 (PAM) 被确定为全基因组转录效应的主要驱动因素.
- 这些影响影响了与细菌防御和代谢相关的基因.
- 在小鼠败血症模型中,全基因组的转录变化与改变的毒性无关.
- 具有单一导向RNA系统的催化无活性的dCas9有效地抑制了特定的GBS基因转录,而没有脱效应.
结论:
- GBS Cas9对转录的影响主要是由于非特异性DNA扫描,而不是其核酶活性.
- 催化不活的dCas9系统为研究GBS基因功能提供了精确的方法.
- 这种工具有潜力用于调查GBS病变发生过程中的必需基因和非必需基因.
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