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通过工程 Cas9-NG 扩展 PAM 可读性的能量学

Shreya Bhattacharya¹, Priyadarshi Satpati¹

¹Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.

Journal of chemical information and modeling

|March 27, 2025

概括

具有七种突变的工程 Cas9-NG 通过防水相互作用和改进的静电接触稳定非同源 PAM 复合体,从而增强原体空间体相邻动机 (PAM) 识别,从而实现更广泛的 DNA 向.

科学领域:

生物化学和分子生物学
结构生物学结构生物学
计算化学计算化学

背景情况:

CRISPR-Cas9系统是一种强大的基因编辑工具,但其准特异性受到原空间器相邻动机 (PAM) 序列的限制.
工程 Cas9 变体,如 Cas9-NG,已被开发用于扩展 PAM 识别,但这种增强可读性的能量基础尚未完全理解.

研究的目的:

为了阐明Cas9-NG中七种特定突变的能量贡献,这些突变可以增强原体空间体相邻动机 (PAM) 的识别.
了解Cas9-NG与野生型SpCas9.9相比观察到的改变DNA裂变活性和更广泛的PAM识别的热力学基础.

主要方法:

使用SpCas9:sgRNA:dsDNA预催化复合物的X射线结构作为结构模板.
进行了严格的炼化模拟 (采样~53μs) 以计算每个七种突变的PAM结合亲和度 (ΔΔG) 的变化.
分析了SpCas9和DNA之间的相互作用能量,以将突变类型和位置与结合亲和关系相关联.

主要成果:

发现R1335V突变通过破坏关键的PAM相互作用来破坏DNA结合的稳定性.
四种突变 (E1219F,D1135V,L1111R和T1337R) 通过引入非基因特异性相互作用并提高PAM可读性,减轻了R1335V的破坏效应.
疏水替代物 (E1219F,D1135V) 特别有效,不包括用于加强静电相互作用的溶剂,并将非同源PAM复合物的稳定性增加2-5kcal/mol.

结论:

稳定Cas9-NG:非同源PAM复合体,由突变诱导的溶解和增强的静电相互作用在疏水口袋驱动,使更广泛的PAM识别.
氨基酸突变的位置对于塑造相互作用能量和改善非正规PAM序列的识别至关重要.
这些发现为合理设计SpCas9突变物提供了基础,这些突变物具有改进和定制的PAM识别能力,用于基因编辑应用.

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