Enhancing CRISPR/Cas-mediated detection of nucleic acids using PNIPAM-based reporters.

Currently most conventional reporters in CRISPR/Cas system, including fluorophore-quencher (FQ) and magnetic bead (MB)-based reporters, encounter limitations in terms of sensitivity and compatibility. To overcome these challenges, we developed novel reporters for CRISPR/Cas systems based on thermo-responsive poly(N-isopropylacrylamide) (PNIPAM). Below the lower critical solution temperature (LCST), PNIPAM-based reporters exhibited a liquid state and can be cleaved by Cas proteins in a homogeneous reaction, preserving function and structure of Cas proteins while effectively accelerating the reaction kinetics. Based on this, we designed three dual-enzyme amplification strategies for ultra-sensitive RNA detection, where RNA-activated LbuCas13a cleaved PNIPAM-based reporters to release enzymes (HRP or ALP) or Cas12a activators as the first amplification, followed by thermal separation to initiate secondary enzymatic amplification. Using SARS-CoV-2 RNA as a model target, these strategies achieved a limit of detection (LOD) as low as ∼1 fM, representing a 100-fold improvement over the traditional CRISPR/Cas13a system, while its excellent practical applicability was validated by spiked recovery assay and specific analysis. Overall, this work proposed novel PNIPAM-based reporters that not only could be applied to various individual CRISPR/Cas systems, but also enable integration with downstream amplification steps through their thermal separation properties, advancing the development of next-generation CRISPR/Cas-based molecular diagnostic tools.