Sun Yat-sen University Nanchang Research Institution, Nanchang, Jiangxi 330200, China.
Department of Gynecological Oncology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, China.
Cell Rep Methods. 2023 Apr 5;3(4):100444. doi: 10.1016/j.crmeth.2023.100444. eCollection 2023 Apr 24.
CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes a TTNA protospacer adjacent motif (PAM) distinct from the canonical TTTV PAM but with equivalent cleavage ability and specificity. These features significantly broadened the targeting scope of Cas12a family. Furthermore, we developed a sensitive, accurate, and rapid human papillomavirus (HPV) 16/18 gene detection platform based on LtCas12a DNA endonuclease-targeted CRISPR reporter (DETECTR) and lateral flow assay (LFA). LtCas12a showed comparable sensitivity to quantitative polymerase chain reaction (qPCR) and no cross-reaction with 13 other high-risk HPV genotypes in detecting the HPV16/18 L1 gene. Taken together, LtCas12a can broaden the applications of the CRISPR-Cas12a family and serve as a promising next-generation tool for therapeutic application and molecular diagnosis.
CRISPR 相关（Cas）核酸酶是基因编辑的多功能工具。 Cas12a 具有几个优点，包括只需要单链向导 RNA 和基因编辑的高保真度。在这里，我们测试了来自人肠道样本的三种 Cas12a 同源物，并鉴定出一种 LtCas12a，它利用与经典 TTTV PAM 不同的 TTNA 前导序列相邻基序（PAM），但具有等效的切割能力和特异性。这些特征显著拓宽了 Cas12a 家族的靶向范围。此外，我们基于 LtCas12a DNA 内切酶靶向 CRISPR 报告（DETECTR）和侧流分析（LFA）开发了一种灵敏、准确、快速的人乳头瘤病毒（HPV）16/18 基因检测平台。 LtCas12a 在检测 HPV16/18 L1 基因时，与定量聚合酶链反应（qPCR）具有可比性的灵敏度，并且与 13 种其他高危 HPV 基因型无交叉反应。综上所述，LtCas12a 可以拓宽 CRISPR-Cas12a 家族的应用，并作为一种有前途的下一代治疗应用和分子诊断工具。
