CRISPR/Cas12a-Responsive Hydrogels for Conjugation-Free and Universal Indicator Release in Colorimetric Detection

Recent advances have demonstrated the significant potential and advantages to repurpose existing point-of-care reactions/devices to realize portable detection of nonoriginal targets, e.g., pathogen genes. However, pursuing this aim usually requires protein indicator-nucleic acid conjugation via a covalent bond, which may bring drawbacks such as high cost, complicated procedure, and annoying component rebuilding. Herein, we developed a conjugation-free, effective, and universal detection platform called CRIs-gel (CRISPR/Cas12a-Responsive Indicators@RCA hydrogels). Various protein indicators are pre-encapsulated into the hydrogels made of effective and high-yield rolling circle amplification (RCA). Upon a targeting sequence binding with its antisense crRNA, CRISPR/Cas12a starts its trans-cleavage activity to crush the hydrogel, which may directly release the indicator for downstream readout. Two proteins, amylase (GA) and human chorionic gonadotropin (hCG), are successfully used as model indicators to trigger the downstream amylum-I₂ color change and pregnancy test strip response. After coupling with upstream isothermal nucleic acid amplification, both portable readouts may detect as few as 2 copies/μL genetic sequences of influenza A virus (FluA), human papilloma virus (HPV), SARS-CoV-2, and influenza B virus (FluB). This conjugation-free CRIs-gel platform is thus simple, sensitive, and universal and can provide innovative insights for portable point-of-care testing (POCT) development.