Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China.
Department of Dermatovenereology, Tianjin Medical University General Hospital, Tianjin 300052, China.
Theranostics. 2023 Sep 25;13(15):5305-5321. doi: 10.7150/thno.84429. eCollection 2023.
The CRISPR/Cas13a system offers the advantages of rapidity, precision, high sensitivity, and programmability as a molecular diagnostic tool for critical illnesses. One of the salient features of CRISPR/Cas13a-based bioassays is its ability to recognize and cleave the target RNA specifically. Simple and efficient approaches for RNA manipulation would enrich our knowledge of disease-linked gene expression patterns and provide insights into their involvement in the underlying pathomechanism. However, only a few studies reported the Cas13a-based reporter system for molecular diagnoses. A tiled crRNA pool targeting a particular RNA transcript was generated, and the optimally potential crRNA candidates were selected using bioinformatics modeling and biological validation methods. For imaging assessment of the anti-GBM effectiveness, we exploited a human GBM patient-derived xenograft model in nude mice. The most efficient crRNA sequence with a substantial cleavage impact on the target RNA as well as a potent collateral cleavage effect, was selected. In the xenografted GBM rodent model, the Cas13a-based reporter system enabled us imaging of the tumor growth. Furthermore, systemic treatments using this approach slowed tumor progression and increased the overall survival time in mice. Our work demonstrated the clinical potential of a Cas13a-based imaging method for the targeted degradation of specific RNAs in glioma cells, and suggested the feasibility of a tailored approach like Cas13a for the modulation of diagnosis and treatment options in glioma.
CRISPR/Cas13a 系统作为一种用于重症疾病的分子诊断工具，具有快速、精确、高灵敏度和可编程性等优点。基于 CRISPR/Cas13a 的生物测定法的一个显著特点是其能够特异性识别和切割靶 RNA。简单高效的 RNA 操作方法将丰富我们对疾病相关基因表达模式的认识，并深入了解它们在潜在病理机制中的作用。然而，只有少数研究报道了基于 Cas13a 的报告系统用于分子诊断。生成了针对特定 RNA 转录本的平铺 crRNA 池，并使用生物信息学建模和生物学验证方法选择最佳的潜在 crRNA 候选物。为了评估抗 GBM 的效果，我们在裸鼠中利用人 GBM 患者来源的异种移植模型进行了成像评估。选择了对靶 RNA 具有显著切割作用和有效旁切作用的最有效 crRNA 序列。在移植的 GBM 啮齿动物模型中，基于 Cas13a 的报告系统使我们能够对肿瘤生长进行成像。此外，使用这种方法进行系统治疗可减缓肿瘤进展并延长小鼠的总生存时间。我们的工作证明了基于 Cas13a 的成像方法在靶向降解神经胶质瘤细胞中特定 RNA 方面的临床潜力，并表明了 Cas13a 等定制方法在调节神经胶质瘤诊断和治疗选择方面的可行性。
