The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2′-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.

CRISPR 相关核酸酶 Cas9 的脱靶活性是治疗性基因组编辑应用的潜在问题。尽管已经设计了高保真 Cas9 变体，但它们表现出不同的效率并具有残留的脱靶效应，限制了它们的适用性。在这里，我们展示了 CRISPR 混合 RNA-DNA (chRDNA) 指南提供了一种有效的方法来增加 Cas9 特异性，同时保留目标编辑活动。在原代人类 T 细胞中的多个基因组靶标中，我们表明 2'-脱氧核苷酸 (dnt) 定位以靶标依赖性方式影响向导活性和特异性，这可用于设计 chRDNA 向导，从而显着降低脱靶效应。DNA 结合的 Cas9-chRDNA 复合物的晶体结构揭示了扭曲的导向-靶标双链几何形状和 Cas9 构象的变构调节。这些结构效应通过扰乱 DNA 杂交和调节 Cas9 激活动力学以不利于脱靶底物的结合和裂解来增加特异性。总的来说，这些结果为在临床应用中使用定制的 chRDNA 铺平了道路。