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