通过SpyCas9的RuvC活动部位的差异二元金属结合有助于非特异性DNA裂变
Sydney N Newsom1, Duen-Shian Wang2, Saadi Rostami1
1Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, The University of Oklahoma, Norman, Oklahoma, USA.
The CRISPR journal
|December 18, 2023
概括
研究人员通过修改Streptococcus pyogenes Cas9 (SpyCas9) 核酶来设计了一个更安全的CRISPR基因编辑工具. 这种变异减少了不必要的DNA裂变,提高了基因编辑应用的精度.
科学领域:
- 分子生物学分子生物学
- 生物化学 生化学
- 生物技术是生物技术.
背景情况:
- 集群定期间隔的短巴林德罗姆重复-CRISPR相关 (CRISPR-Cas) 系统为细菌和古生物提供了对移动遗传元素 (MGE) 的适应性免疫力.
- 克里斯普尔-卡斯系统利用克里斯普尔RNAs (crRNAs) 和卡斯核酶进行序列特定的DNA裂变,这是适用于基因编辑应用的机制.
- 基因编辑的安全问题包括非目标裂变和指导RNA (gRNA) 独立的DNA裂变由核酶,如Streptococcus pyogenes Cas9 (SpyCas9).
研究的目的:
- 为了设计一种SpyCas9的变体,减少或消除无gRNADNA裂变活性.
- 为了提高SpyCas9在基因编辑应用中的特异性和安全性.
- 通过SpyCas9.9来研究gRNA自由DNA裂变中的阴离子辅因子的作用.
主要方法:
- 用局部定向突变生成来创建一个SpyCas9变体,在RuvC活性部位中用H982A替代.
- 进行了体外测试,以测量在不同双价金属 (Mn2+和Mg2+) 存在时的gRNA-free DNA裂变活性.
- 机械分子动力学模拟被用来分析基离子依赖DNA裂变的结构基础.
主要成果:
- 在存在Mn2+的情况下,SpyCas9 H982A变体表现出显著降低的无gRNADNA裂变活性,大约是野生型SpyCas9的167倍.
- 分子动力学模拟显示,Mn2+在野生型SpyCas9中促进了无gRNA的DNA分裂能力状态,这种状态被H982A替代破坏.
- 替代H982A特别针对Mn2+依赖的gRNA无DNA裂变,而Mg2+依赖的裂变活动基本上不受影响.
结论:
- H982A替代有效地消除了依赖Mn2+的gRNA无DNA裂变,从而导致更安全,更有选择性的SpyCas9变种用于基因编辑.
- 调节阴离子:蛋白相互作用为工程改进和更安全的CRISPR-Cas基因编辑工具提供了可行的策略.
- 这项研究提供了对阴离子依赖DNA裂变的机制性理解,并为开发下一代基因编辑技术提供了途径.
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