An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

• Main Authors: Jiaojiao Gong, Lijuan Kan, Xiuming Zhang, Ying He, Jiaqiang Pan, Liping Zhao, Qianyun Li, Menghao Liu, Jie Tian, Sili Lin, Zhouyu Lu, Liang Xue, Chaojun Wang, Guanghui Tang
• Format: Article
• Language: English
• Published: KeAi Communications Co., Ltd. 2021-12-01
• Series: Bioactive Materials
• Subjects: CRISPR-Cas12a; Onepot; Visual detection
• Online Access: http://www.sciencedirect.com/science/article/pii/S2452199X21002255

CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction.Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal. This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection.Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.