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Hiroshi Nishimasu1, Xi Shi2,3, Soh Ishiguro4,5,6

  • 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. nisimasu@bs.s.u-tokyo.ac.jp nureki@bs.s.u-tokyo.ac.jp.

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概括

科学家设计了一种Cas9酶变体 (SpCas9-NG),以识别放松的NG原空间器相邻动机 (PAM). 这扩大了基因组编辑能力,通过准人类细胞中以前无法访问的基因组位置.

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科学领域:

  • 分子生物学
  • 基因组学
  • 生物化学

背景情况:

  • Cas9酶是基因组编辑的一个关键工具,使得有针对性的DNA分裂成为可能.
  • 通常使用的Streptococcus pyogenes Cas9 (SpCas9) 酶因其对特定的NGG原体隔离基因 (PAM) 的要求而受到限制.
  • 这种NGG PAM特异性限制了可以针对编辑的基因组位点的范围.

研究的目的:

  • 设计一种具有放松 PAM 识别能力的 SpCas9 变种.
  • 通过严格的NGG PAM要求克服野生类型SpCas9的限制.
  • 扩大基于CRISPR的基因组编辑目标基因组位置的范围.

主要方法:

  • 合理设计SpCas9酶以创建具有改变PAM特异性的变体 (SpCas9-NG).
  • 通过X射线晶体学来确定NG PAM识别的结构基础.
  • 在人体细胞中进行功能性测试,以评估SpCas9-NG在NG PAM部位诱导indels的能力.
  • 用于测试NG PAM位点的基编辑能力,将SpCas9-NG与激活诱导的cytidine deaminase (AID) 融合.

主要成果:

  • 设计成功的SpCas9-NG变种能够识别放松的NG PAM.
  • 晶体结构揭示了SpCas9-NG通过改变基因特异性相互作用来适应NG PAM.
  • 在诱导人体细胞内源性NGPAM位点的插入/删除 (indels) 中,SpCas9-NG被证明是有效的.
  • 与AID融合的SpCas9-NG促进了人体细胞中NG PAM位点的向C-T转化.

结论:

  • 基于Cas9的基因组编辑技术取得了重大进展.
  • 设计的SpCas9-NG扩大了准范围,使以前无法访问的基因组位置能够进行编辑.
  • 与AID的SpCas9-NG融合为NG PAM站点提供了精确基因编辑的多功能工具,增强了基因组工程应用.