Single-molecule assay for highly sensitive SARS-CoV-2 gene detection based on CRISPR-Cas12a

Abstract

A trans-cleavage activity of some CRISPR-associated (Cas) proteins including Cas12a and Cas 13a that non-specifically cleaves single-stranded DNA or RNA, has been wildly used to develop sensitive gene detection methods because it is a multiple-turnover enzymatic process, only activated via guide RNA-target gene pairing. Combined with a pre-amplification step, CRISPR-Cas12a-based gene detection methods achieved even attomolar detection limits. However, the pre-amplification step often accompanies the tedious design of primers and inevitable base substitution errors by DNA polymerases, which is not suitable for quantitative analysis. Here, we report a single-molecule assay for ultrasensitive and quantitative gene detection utilizing the trans-cleavage activity of Cas12a without pre-amplification. To monitor the trans-cleavage of Cas12a at the single-molecule level, we designed a hairpin DNA probe with a stem-loop structure where the single-stranded DNA loop can be cleaved via the trans-cleavage of Cas12a, labeled fluorophore and quencher pairs at the end of the stem and immobilized it at the surface. Only in the existence of target gene, the trans-cleavage of Cas12a is activated, cleaving the loop of the hairpin DNA probe, making the quencher leave from the surface, and resulting in the appearance of a fluorescent spot. In other words, the number of appearing fluorescent spots reflects the amount of target gene quantitatively. After optimization of various experimental conditions such as different Cas12a orthologs, monovalent and divalent ion concentration in the reaction buffer, reaction temperature and the mixing ratio of Cas12a to guide RNA, we successfully achieved to detect the E gene of SARS-CoV-2 down to sub picomolar range without any pre-amplification step.