位于 PAM 下游的 Cas12a 和 dsDNA 之间的非特异性相互作用介导目标搜索并协助 AsCas12a 进行 DNA 切割,Chemical Science

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位于 PAM 下游的 Cas12a 和 dsDNA 之间的非特异性相互作用介导目标搜索并协助 AsCas12a 进行 DNA 切割
Chemical Science ( IF 7.6 ) Pub Date : 2023-03-10 , DOI: 10.1039/d2sc05463a
Ruirui Sun _{1,

2} , Yuqian Zhao _{1,

3} , Wenjuan Wang ₄ , Jun-Jie Gogo Liu _{1,

3} , Chunlai Chen _{1,

2}

Affiliation

Cas12a 是最常用于基因组编辑和基因调控的 Cas 蛋白之一。Cas12a 实现其功能的第一个关键步骤是在众多非特异性和脱靶位点中搜索其目标。Cas12a 利用沿 dsDNA 轮廓的一维扩散来有效地搜索其目标。然而，由于缺乏瞬时扩散复合物的结构信息，介导 Cas12a 一维扩散的残基是未知的。在这里，结合单分子和整体分析，我们发现 PAM 下游的 Cas12a 和 dsDNA 之间的非特异性相互作用导致 PAM 位点侧翼的 Cas12a 的不对称目标搜索区域，这引导我们在 REC2 域中识别出富含正电荷的 α 螺旋作为保守元素，以促进 AsCas12a、LbCas12a 和 FnCas12a 的一维扩散驱动目标搜索。此外，此 alpha 螺旋有助于 AsCas12a 的目标切割过程通过稳定分裂状态。因此，中和该螺旋内的正电荷不仅会显着减慢目标搜索速度，还会增强 AsCas12a在体外和活细胞中的特异性。当介导 SpCas9 扩散的残基发生突变时，会检测到类似的行为。因此，介导 dsDNA 扩散的工程残基是优化和丰富多功能 CRISPR-Cas 工具箱的新途径。

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Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage

Cas12a is one of the most commonly used Cas proteins for genome editing and gene regulation. The first key step for Cas12a to fulfill its function is to search for its target among numerous nonspecific and off-target sites. Cas12a utilizes one-dimensional diffusion along the contour of dsDNA to efficiently search for its target. However, due to a lack of structural information of the transient diffusing complex, the residues mediating the one-dimensional diffusion of Cas12a are unknown. Here, combining single-molecule and ensemble assays, we found that nonspecific interactions between Cas12a and dsDNA at the PAM downstream cause asymmetric target search regions of Cas12a flanking the PAM site, which guided us to identify a positive-charge-enriched alpha helix in the REC2 domain serving as a conserved element to facilitate one-dimensional diffusion-driven target search of AsCas12a, LbCas12a and FnCas12a. In addition, this alpha helix assists the target cleavage process of AsCas12a via stabilizing the cleavage states. Thus, neutralizing positive charges within this helix not only significantly slows target search but also enhances the specificity of AsCas12a both in vitro and in living cells. Similar behaviors are detected when residues mediating diffusion of SpCas9 are mutated. Thus, engineering residues mediating diffusion on dsDNA is a new avenue to optimize and enrich the versatile CRISPR-Cas toolbox.

更新日期：2023-03-10

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