Jiang Mingjun, Ye Yanzhen, Li Juan
State Key Laboratory of Natural and Biomimetic Drugs, Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China.
SLAS Technol. 2021 Feb;26(1):92-102. doi: 10.1177/2472630320922813. Epub 2020 Jun 3.
The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a widely used genome-editing tool with great clinical potential. However, its application is limited because of low editing efficiency of some target sequences and off-target effects. As this system contains only the Cas9 protein and a single-guide RNA (sgRNA; engineered from crRNA and tracrRNA), the structure and function of these components should be studied in detail to address the current clinical needs. Consequently, we investigated the structural and sequence features of the core hairpin (the first stem loop of sgRNA) of SpCas9 sgRNA. We showed that the core hairpin structure of sgRNA is essential for SpCas9/sgRNA-mediated DNA cleavage and that the internal loop structure in the core hairpin plays a vital role in target DNA cleavage. We observed that the root stem structure within the core hairpin preferentially forms Watson-Crick base pairs and should be of a specific length to maintain an appropriate spatial conformation for Cas9 binding. However, the length of the leaf stem structure of the core hairpin is flexible, having a variable nucleotide composition. Furthermore, extension of the leaf stem structure enhances the DNA cleavage activity of the Cas9/sgRNA complex, and this could be used to enhance the efficiency of gene editing. These observations provide insight into the sgRNA/Cas9 interaction, indicating that sgRNA modification could be a strategy for improved DNA editing efficiency, and optimized sgRNA can be further used for genome-wide functional screening and clinical application.
成簇规律间隔短回文重复序列（CRISPR）/Cas9系统是一种广泛应用且具有巨大临床潜力的基因组编辑工具。然而，由于一些靶序列的编辑效率低以及脱靶效应，其应用受到限制。由于该系统仅包含Cas9蛋白和单导向RNA（sgRNA；由crRNA和tracrRNA改造而来），因此应详细研究这些组件的结构和功能，以满足当前的临床需求。因此，我们研究了SpCas9 sgRNA核心发夹结构（sgRNA的第一个茎环）的结构和序列特征。我们发现，sgRNA的核心发夹结构对于SpCas9/sgRNA介导的DNA切割至关重要，并且核心发夹中的内环结构在靶DNA切割中起着关键作用。我们观察到，核心发夹内的根茎结构优先形成沃森-克里克碱基对，并且应为特定长度，以维持与Cas9结合的合适空间构象。然而，核心发夹的叶茎结构长度具有灵活性，核苷酸组成可变。此外，叶茎结构的延伸增强了Cas9/sgRNA复合物的DNA切割活性，这可用于提高基因编辑效率。这些观察结果为sgRNA/Cas9相互作用提供了深入了解，表明sgRNA修饰可能是提高DNA编辑效率的一种策略，优化后的sgRNA可进一步用于全基因组功能筛选和临床应用。
