一个紧的CjCas9变体的持续定向演变,具有广泛的PAM兼容性
Lukas Schmidheini1,2, Nicolas Mathis1, Kim Fabiano Marquart1,2
1Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Nature chemical biology
|September 21, 2023
概括
在CRISPR-Cas9基因组工程方面取得了进展,evoCjCas9是一种Campylobacter jejuni Cas9 (CjCas9) 的变种. 这种增强的CRISPR工具扩大了准范围,并改善了对遗传疾病的体内基因编辑.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物技术是生物技术.
背景情况:
- 克里斯普尔-Cas9技术为基因疾病的纠正提供了潜力.
- 局限性包括受限的原体空间邻动机 (PAM) 识别和大Cas9大小阻碍体内输送.
研究的目的:
- 使用定向进化扩大Campylobacter jejuni Cas9 (CjCas9) 的PAM兼容性.
- 为增强基因组工程应用开发一个更小,更通用的Cas9变体.
主要方法:
- 菌体辅助的连续定向进化被用来设计CjCas9.
- 进化的变体,evoCjCas9,以PAM特异性和核酶活性为特征.
主要成果:
- 这种evoCjCas9变体识别了N4AH和N5HA PAM序列,与野生型CjCas9.9相比,增加了基因组准频率的十倍.
- evoCjCas9表现出增强的核酶活性和与PAM放松的SpCas9变体相比的编辑速率.
- 小的evoCjCas9基编辑器通过单个腺相关病毒载体促进了有效的A-to-G和C-to-T过渡.
结论:
- 设计的evoCjCas9显著扩大了CRISPR-Cas9的准范围和效率.
- 它的小尺寸和广泛的PAM兼容性使其能够用于基因疾病治疗的基础和主要编辑等高级应用.
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