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Updated: Jul 25, 2025

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Published on: June 16, 2017

经过工程改造的Staphylococcus auricularis Cas9具有高保真性

Nan Wei^1,2, Lu Shang^1,2, Jing Liu^1,2

¹Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

|June 30, 2023

概括

设计的CRISPR-Cas9变体SauriCas9-HF1和SauriCas9-HF2显著提高了基因编辑的特异性. 这些改进的工具减少了非目标效应,扩大了CRISPR在研究和治疗中的应用.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
生物技术是生物技术.

背景情况:

CRISPR-Cas9是一种强大的基因编辑工具,但非目标效应限制了其临床使用.
耳性葡萄球菌Cas9 (SauriCas9) 是一个小的,活跃的Cas9 Ortholog,具有灵活的5'-NNGG-3' PAM识别.
增强忠实性SaCas9 (efSaCas9) 通过单个突变显示出更好的特异性.

研究的目的:

通过应用efSaCas9.9的突变来设计具有提高特异性的SauriCas9变体.
评估工程SauriCas9变体的准特异性和非准效应.

主要方法:

在SauriCas9和SaCas9.9之间的蛋白质序列对齐.
具有特定突变的SauriCas9变体 (SauriCas9-HF1,SauriCas9-HF2) 的工程.
评估使用目标深度测序和 GUIDE-seq. 的目标特异性的评估.

主要成果:

两种设计的SauriCas9变种,即SauriCas9-HF1 (N269D) 和SauriCas9-HF2 (D270N),已经成功创建.
与野生型SauriCas9.9相比,这两种变体都显示出明显改善的准特异性.
SauriCas9-HF2显示出非目标效应的显著减少 (某些部位的改善为61.6至111.9倍).

结论:

设计的SauriCas9变体SauriCas9-HF1和SauriCas9-HF2增强了CRISPR-Cas9的特异性.
这些变异减少了非目标突变,使得它们对精确的基因组编辑有价值.
开发的SauriCas9变种扩展了CRISPR工具包,用于研究和治疗应用.

关键词:

克里斯普尔-Cas9是什么意思耳性葡萄球菌Cas9 (SauriCas9) 是一种高保真度的高保真度显示器非目标效应的非目标效应

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