Cas12a-based primer production enables isothermal amplification for nucleic acid detection

Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated Cas proteins have been employed as innovative tools for biomolecule detection. However, the functionality of the Cas protein is limited by the RNA-guided nuclease that cleaves pre-amplified products to generate fluorescence signals. As such, we proposed a strategy to use the Cas12a protein for active primer production and as an initiator for subsequent isothermal amplification. Target nucleic acids activate Cas12a to cleave the DNA component of RNA-DNA chimera primers with a 3′ phosphate group, converting inactive RNA-DNA chimera primers into active ones. We then used two isothermal amplification methods based on Klenow DNA polymerase/T7 RNA polymerase and terminal deoxynucleotidyl transferase to generate fluorescence and colorimetric signals, respectively. The Salmonella enterotoxin (stn) gene was effectively amplified with satisfactory sensitivity and selectivity even in real samples such as drinking water and human serum, paving the way for Cas12a-derived active primers that may be useful in diverse detection platforms.