Terminal Chemical\nModifications of crRNAs Enable Improvement\nin the Performance of CRISPR-Cas for Point-of-Care Nucleic Acid Detection

CRISPR-Cas systems, harnessing their precise nucleic\nacid recognition\nvia CRISPR RNA (crRNA), offer promise for the accurate testing of\nnucleic acids in the field. However, the inherent susceptibility of\ncrRNA to degradation poses challenges for accurate detection in low-resource\nsettings. Here, we utilized the chemically modified crRNA for the\nCRISPR-Cas-based assay (CM-CRISPR). We found that the extension and\nchemical modification to crRNA significantly enhanced the <i>trans</i>-cleavage activity of LbCas12a. The chemically modified\ncrRNA was resistant to degradation, and CM-CRISPR showed superior\ndetection capability in complex environments. CM-CRISPR could be combined\nwith recombinase polymerase amplification (RPA) and applied in a droplet\ndigital platform, enabling attomolar-level sensitivity. We also developed\na portable and automated device for a digital CRISPR assay, which\nis amenable to point-of-care testing (POCT). The extraction-free procedure\nwas integrated with this assay to streamline the workflow, and clinical\nsamples were successfully detected. This work finds a simple and efficient\nway to improve the performance of CRISPR-Cas and develops a portable\nplatform for POCT, representing a significant advance toward practical\napplications of CRISPR-based diagnostics.