Kanazhevskaya Lyubov Yu, Zhdanova Polina V, Chernonosov Alexander A, Koval Vladimir V
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia.
Department of Natural Sciences, Novosibirsk State University, 630090 Novosibirsk, Russia.
Biochem Biophys Rep. 2025 Jul 5;43:102134. doi: 10.1016/j.bbrep.2025.102134. eCollection 2025 Sep.
The in vivo editing of genetic information necessitates tools of unprecedented accuracy. CRISPR-Cas-based systems have emerged as leading technologies for precisely targeting the genome. The Cas9 endonuclease derived from is the most commonly used instrument for targeted DNA cleavage. The development of engineered and chimeric Cas9 variants with enhanced activity and specificity has enabled not only the simple knockout of target genes but also the sophisticated engineering of the epigenome. This advancement has broadened the potential applications of CRISPR-Cas9 technology for the treatment of various disorders characterized by a combination of mutations, deletions, and duplications in the coding and non-coding regions of the genome. The inherent simplicity and predictability of the CRISPR-Cas9 targeting mechanism have led to an explosive growth in the development of prototype gene-editing drugs. However, their therapeutic application is still challenged by potential off-target effects. The erroneous editing of tumor suppressors and oncogenes could lead to adverse outcomes that mitigate the benefits of CRISPR therapy. The evolution of DNA-targeting technologies requires a comprehensive understanding of the mechanisms underlying CRISPR-Cas9 off-target binding and cleavage. The use of massive libraries of DNA targets and guide RNAs, coupled with high-throughput sequencing, contributes significantly to the analysis of mismatch tolerance. Nevertheless, the detection of ultra-low levels of off-target activity is hindered by the sensitivity limitations of current technologies. This review focuses on the mechanisms responsible for off-target interactions during CRISPR-Cas9-mediated gene editing. We discuss the influence of various factors, including nucleotide context, enzyme concentration, guide RNA structure, and the energetics of the RNA-DNA hybrid on mismatch tolerance in vitro and in vivo. Recent advances in the development of technologies for predicting off-target effects are briefly summarized. Particular emphasis is placed on the role of the Cas9 protein structure in the allosteric regulation of the specific and non-specific activity of the Cas9-sgRNA complex.
体内遗传信息编辑需要具有前所未有的准确性的工具。基于CRISPR-Cas的系统已成为精确靶向基因组的领先技术。源自酿脓链球菌的Cas9核酸内切酶是最常用于靶向DNA切割的工具。具有增强活性和特异性的工程化和嵌合Cas9变体的开发不仅实现了靶基因的简单敲除，还实现了表观基因组的复杂工程改造。这一进展拓宽了CRISPR-Cas9技术在治疗各种由基因组编码和非编码区域的突变、缺失和重复组合所表征的疾病方面的潜在应用。CRISPR-Cas9靶向机制固有的简单性和可预测性导致了原型基因编辑药物开发的爆炸式增长。然而，它们的治疗应用仍然受到潜在脱靶效应的挑战。肿瘤抑制因子和癌基因的错误编辑可能导致不良后果，从而削弱CRISPR治疗的益处。DNA靶向技术的发展需要全面了解CRISPR-Cas9脱靶结合和切割的潜在机制。使用大量的DNA靶标和引导RNA文库，结合高通量测序，对错配耐受性分析有很大贡献。然而，当前技术的灵敏度限制阻碍了超低水平脱靶活性的检测。本综述重点关注CRISPR-Cas9介导的基因编辑过程中脱靶相互作用的机制。我们讨论了各种因素的影响，包括核苷酸背景、酶浓度、引导RNA结构以及RNA-DNA杂交体的能量学对体外和体内错配耐受性的影响。简要总结了预测脱靶效应技术开发的最新进展。特别强调了Cas9蛋白结构在Cas9-sgRNA复合物特异性和非特异性活性变构调节中的作用。
