CRISPR-Cas12a exhibits metal-dependent specificity switching.
CRISPR-Cas12a exhibits metal-dependent specificity switching.
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CRISPR-Cas12a 表现出金属依赖性特异性转换。
DOI：
10.1101/2023.11.29.569287
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Sashital,DipaliG
中科院分区：
文献类型：
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作者：
Nguyen,GiangT;Schelling,MichaelA;Buscher,KathrynA;Sritharan,Aneisha;Sashital,DipaliG
Cas 12 a是V-A型CRISPR-Cas系统的免疫效应子，已被用于基因组编辑和其他生物技术工具。Cas 12 a的特异性一直是体外和基因组编辑实验中广泛研究的主题。然而，体外研究通常在高镁离子浓度下进行，这与细胞中存在的游离Mg 2+浓度不一致。通过分析Cas 12 a直系同源物在一系列Mg 2+浓度下的特异性，我们发现Cas 12 a根据金属离子浓度改变其特异性。降低Mg 2+浓度减少了由种子失配引起的解理缺陷，同时增加了由PAM远端失配引起的缺陷。我们表明，Cas 12 a可以在低Mg 2+浓度下更快地结合种子突变体靶标，从而导致更快的切割。相比之下，PAM-远端错配在低Mg 2+浓度下形成Cas 12 a-靶复合物后导致切割的实质性缺陷。我们观察到三种直系同源物之间Cas 12 a特异性转换的差异，这导致噬菌体从Cas 12 a介导的免疫中逃逸的途径发生变化。总的来说，我们的结果揭示了生理金属离子条件对在不同细胞环境中使用的Cas效应物的特异性的重要性。
Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation bothin vitroand in genome editing experiments. However,in vitrostudies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.