一个Cas12i核酶的结构引导工程解锁了几乎没有PAM的基因组编辑
Qitong Chen1, Hanlin Gou1, Chao Xu1
1Institute of Advanced Biotechnology, Institute of Homeostatic Medicine, and School of Medicine, Southern University of Science and Technology, Shenzhen, China.
Advanced science (Weinheim, Baden-Wurttemberg, Germany)
|January 14, 2026
概括
研究人员设计了CRISPR-Cas12i核酶 (SF01变体),以克服原体空间器相邻动机 (PAM) 的限制. 这一突破将基因组编辑的可访问性扩展到超过25%的基因组,使新的治疗和研究应用成为可能.
科学领域:
- 分子生物学分子生物学
- 基因编辑技术的技术
背景情况:
- 克里斯普尔-Cas核酶是强大的基因组编辑工具,但受到严格的原生空间器相邻动机 (PAM) 识别序列的限制.
- 这种PAM特异性限制了治疗和研究应用的基因组内的可访问位置.
研究的目的:
- 为了设计一个紧的Cas12i核酶 (SF01),具有放松的PAM特异性.
- 扩大CRISPR-Cas基因组编辑工具的目标范围.
主要方法:
- 使用AlphaFold预测模型对SF01核酶进行结构指导工程.
- 在PAM相互作用接口的38个残留物的系统性突变发生.
- 构建和测试具有工程变体的腺基编辑器 (ABE).
主要成果:
- 开发了三种优越的SF01变体 (KR,IKRR,STKRR) 具有放松的PAM特异性,可在5'-NNTN-3'位点进行编辑.
- 实现了可向基因组部分的四倍增长 (超过25%),几乎没有PAM活动.
- 经过工程设计的ABE在内生局部表现出高效编辑 (~80%),目标范围扩大.
- 目标之外的分析证实,尽管目标活动增强,但特异性没有丧失.
结论:
- 工程 SF01 变种通过克服 PAM 约束来显著扩展基因组编辑工具包.
- 这些变异使以前无法访问的基因组编辑应用程序成为可能.
- 增强的特异性和广泛的准范围为分子生物学和基因治疗提供了一个强大的新工具.
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