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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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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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评估和设计用于编程基因组编辑的IscB-ωRNA系统.

Hao Yan1,2, Xiaoqing Tan3, Siyuan Zou1,2

  • 1Department of Chemistry, The University of Chicago, Chicago, IL, USA.

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|July 8, 2024
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概括

研究人员设计了IscB,一个OMEGA RNA (ωRNA) 引导的内核酶,用于在人体细胞中增强体内基因组编辑. 改进的OgeuIscB系统显示了DNA结合和编辑效率的提高,使其成为基因编辑应用的强大工具.

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科学领域:

  • 分子生物学分子生物学
  • 基因组工程是基因组工程.
  • 生物技术是生物技术.

背景情况:

  • IscB是一种OMEGA RNA (ωRNA) 引导的内核酶,是Cas9的进化祖先.
  • 伊斯克B具有紧的尺寸和与Cas9的机械相似性,使其成为体内基因组编辑的吸引力.
  • 野生类型的IscB-ωRNA系统在人体细胞中表现出有限的活性,因此需要改进才能有效编辑基因组.

研究的目的:

  • 提高IscB-ωRNA系统在人类基因组编辑中的活性和效率.
  • 根据IscB内核酶设计一个更强大和更具特异性的基因组编辑工具.

主要方法:

  • 在IscB中引入了八种氨基酸替代,以创建增强的OgeuIscB变体.
  • 使用结构导向方法设计 ωRNA.
  • 在人类细胞的26个目标部位测试了OgeuIscB-ωRNA系统.
  • 分析了DNA结合亲和力,插入删除 (indel) 形成,基数编辑频率和非目标效应.

主要成果:

  • 与野生型IscB相比,OgeuIscB在体外DNA结合亲和力增加了四倍.
  • 在人体细胞中实现了30.4倍的英德尔形成效率的改善.
  • 在多个目标站点实现了高达87.3%的indel和62.2%的基础编辑频率.
  • 确定了一个NARR目标相邻动机 (TAM) 和特定的R环核酸作为目标选择的关键决定因素.

结论:

  • 经过工程设计的OgeuIscB-ωRNA系统在人类基因组编辑方面表现出显著增强的功效和效率.
  • 该系统显示可编程性和足够的特异性用于治疗和研究应用.
  • OgeuIscB-ωRNA代表了对现有的基因组编辑技术的有希望的替代方案.