紧型热爱性Anoxybacillus flavithermus Cas9核酶的表达和功能分析
Anastasiya Matveeva1, Alexander Ryabchenko1, Viktoria Petrova1
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
International journal of molecular sciences
|December 9, 2023
概括
研究人员设计了AnoCas9,这是一种来自Anoxybacillus flavithermus的新型热友性Cas9核酶. 这种紧的CRISPR-Cas系统在37-60°C之间有效运行,扩展基因组工程工具包.
科学领域:
- 分子生物学分子生物学
- 生物技术是生物技术.
- 基因组学就是基因组学.
背景情况:
- 克里斯普尔-卡斯系统是基因组工程和基因治疗的重要工具.
- 热性Cas9核酶由于它们在更高温度下的稳定性而具有独特的优势.
- 现有的热友性Cas9核酶,如GeoCas9和ThermoCas9,具有特定的活动范围和PAM偏好.
研究的目的:
- 为了生成表达AnoCas9的结构,一个来自Anoxybacillus flavithermus的紧的Cas9核酶.
- 描述AnoCas9.9的核酶活性和PAM偏好.
- 探索AnoCas9在基于热稳定CRISPR/Cas的技术中的潜在应用.
主要方法:
- 对Anoxybacillus flavithermus基因组进行生物信息分析,以识别Cas9的正义基因.
- 对AnoCas9表达的基因构造设计和合成.
- 在温度范围 (37-60°C) 中对AnoCas9核酶活性进行了体外表征.
- 确定AnoCas9的原空间体相邻动图 (PAM) 偏好 (5'-NNNNNCDAA-3').
主要成果:
- 成功生成了一个AnoCas9表达式的构造.
- 在37-60°C范围内,AnoCas9表现出核酶活性.
- 阿诺卡斯9显示了对5'-NNNNNCDAA-3'的特定PAM偏好.
- AnoCas9与紧密相关的GeoCas9家族的紧型热友 Cas9 效应器.
结论:
- AnoCas9代表了一种新的,紧的热爱性Cas9核酶.
- 安诺卡斯9的特征性质扩大了可用的卡斯9核酶的范围.
- 在体外应用中,AnoCas9具有潜力,需要热稳定的CRISPR/Cas系统,包括测序库丰富和异位基因特定的异热PCR.
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