来自Flavobacterium psychrophilum的Fp2Cas9,一种冷适应的II-C型CRISPR核酶的功能性表征
Ran Zhao1, Jianqiang Zhu1, Jing Wang1
1Key Laboratory of Aquatic Animal Diseases and Immune Technology of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China.
International journal of molecular sciences
|November 13, 2025
概括
研究人员发现了Fp2Cas9,这是一种来自*Flavobacterium psychrophilum*的冷活性Cas9酶. 这种酶在低温下有效编辑基因组,扩大了在寒冷环境中的CRISPR应用.
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:
- 在CRISPR-Cas9基因组编辑中,需要具有不同温度适应性的酶.
- 低温活性Cas9变种尚未得到充分探索,限制了在水产养殖等低温系统中的应用.
- 描述适应寒冷的Cas9扩展了CRISPR工具包,用于各种生物环境.
研究的目的:
- 为了功能性地表征Fp2Cas9,Flavobacterium psychrophilum*的适应寒冷的II-C型核酶.
- 为了评估Fp2Cas9的DNA裂变效率和PAM在低温下的特异性.
- 评估Fp2Cas9在斑马鱼体内的体内基因组编辑能力.
主要方法:
- 试验室内DNA分裂试验以确定Fp2Cas9活性和PAM偏好 (5'-SNAAAG-3').
- 在5°C和35°C下比较Fp2Cas9和SpCas9的裂变效率.
- 在体内突变的实验使用核局部化的Fp2Cas9变体 (2NLS-Fp2Cas9) 在斑马鱼胚胎准*slc45a2*基因.
主要成果:
- Fp2Cas9证明了高效的双链DNA裂变,具有特定的PAM要求.
- 在5°C时,Fp2Cas9保持了75%的裂变效率,明显高于SpCas9.
- 在斑马鱼中,2NLS-Fp2Cas9诱导了约60%的独立频率和约43%的胚胎中可观察到的表型.
结论:
- Fp2Cas9是一种功能冷适应的Cas9核酶,具有可靠的低温活性.
- 这一发现为冷水生物和其他低温环境中的基因组编辑提供了有价值的新工具.
- Fp2Cas9扩大了基于CRISPR的基因组工程在各种热条件中的适用性.
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