G-triplex: A new type of CRISPR-Cas12a reporter enabling highly sensitive nucleic acid detection
G-triplex: A new type of CRISPR-Cas12a reporter enabling highly sensitive nucleic acid detection
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G-triplex：一种新型 CRISPR-Cas12a 报告基因，可实现高灵敏度核酸检测
DOI：
10.1016/j.bios.2021.113292
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发表时间：
2021-05-12
影响因子：
12.6
通讯作者：
Liu, Maili
中科院分区：
文献类型：
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作者：
Li, Tao;Hu, Rui;Liu, Maili
CRISPR-Cas 12 a（Cpf 1）反式切割ssDNA，这一特征已被广泛用于核酸检测。在此，我们介绍了一种新型的Cas 12 a报告基因，G-三链体（G3），和一种高度灵敏的生物传感器，称为GCRISPR。我们证明了Cas 12 a在约10 min内反式切割G3结构，并且基于切割诱导的高阶结构破坏，G3可以充当优秀的报告基因。G3报告基因将分析灵敏度提高了20倍，能够分别检测低至50 pmol和0.1阿莫尔（一个拷贝/反应）的未扩增和扩增DNA。G-CRISPR已被用于分析27个PCR扩增的HPV感染风险患者样本，基于荧光和侧流检测，两者之间的一致性为100%。与临床结果比较，其总体特异性和敏感性分别为100%和94.7%。这些结果表明，G-CRISPR可以作为一种快速、灵敏和可靠的生物传感器，并可以进一步扩展CRISPR在生物医学诊断中的工具箱。
CRISPR-Cas12a (Cpf1) trans-cleaves ssDNA and this feature has been widely harnessed for nucleic acid detection. Herein, we introduce a new type of Cas12a reporter, G-triplex (G3), and a highly sensitive biosensor termed GCRISPR. We proved that Cas12a trans-cleaves G3 structures in about 10 min and G3 can serve as an excellent reporter based on the cleavage-induced high-order structure disruption. G3 reporter improves the analytical sensitivity up to 20 folds, enabling the detection of unamplified and amplified DNA as low as 50 pmol and 0.1 amol (one copy/reaction), respectively. G-CRISPR has been utilized for the analysis of 27 PCR-amplified patient samples with HPV infection risk based on both fluorescence and lateral flow assays, resulting in 100% concordance between the two. In comparison with the clinical results, it achieved overall specificity and sensitivity of 100% and 94.7%, respectively. These results suggest that G-CRISPR can serve as a rapid, sensitive, and reliable biosensor, and could further expand the CRISPR toolbox in biomedical diagnostics.