State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361102, China.
Nucleic Acids Res. 2023 May 8;51(8):4064-4077. doi: 10.1093/nar/gkad178.
CRISPR (clustered regularly interspaced short palindromic repeats) technology holds tremendous promise for gene regulation and editing. However, precise control of CRISPR editing is essential to overcome its uncontrollable reaction process and excessive activity that leads to off-target editing. To overcome this problem, we engineered a photoswitch on G-quadruplex gRNA (GqRNA) for precisely controlled gene editing and expression by embedding dicationic azobenzene derivatives (AZD++). Our results demonstrated that rational design of the G-quadruplex onto crRNA conferred higher stability and sequence recognition specificity than unmodified single guide (sgRNA). Light-induced isomerization of AZD++ quickly transformed the on state of GqRNA, which facilitated rapid activation of ribonucleoprotein activity for genome editing of on-target sites in cells with excellent editing efficiency. In turn, AZD++-GqRNA promptly refolded to an off state to inhibit genomic cleavage, and limited the generation of off-target effects and by-products. Therefore, the proposed strategy of a photo-reversible modality presents a new opportunity for CRISPR-Cas9 modulation to improve its safety and applicability.
CRISPR（成簇规律间隔短回文重复序列）技术在基因调控和编辑方面具有巨大的应用前景。然而，要克服 CRISPR 编辑过程的不可控性和过度活性，实现精准控制，需要对其进行精确控制，以克服其脱靶编辑。为了解决这个问题，我们在 G-四链体 gRNA（GqRNA）上设计了一种光开关，通过嵌入二价阳离子偶氮苯衍生物（AZD++），实现了精确控制的基因编辑和表达。我们的研究结果表明，将 G-四链体合理地设计到 crRNA 上，比未修饰的单指导 RNA（sgRNA）具有更高的稳定性和序列识别特异性。AZD++的光诱导异构化迅速将 GqRNA 的状态转换为开启状态，从而促进了核糖核蛋白在细胞中对靶位点的基因组编辑活性的快速激活，具有较高的编辑效率。相反，AZD++-GqRNA 迅速折叠回关闭状态，抑制基因组切割，限制了脱靶效应和副产物的产生。因此，所提出的光可逆调控策略为 CRISPR-Cas9 调节提供了新的机会，提高了其安全性和适用性。
