Development of a universal one-pot CRISPR assay based on multifunctional tagged primer eliminating unstable crRNA input and PAM dependency for point-of-care detection of bacterial infections.

The integration of recombinase polymerase amplification (RPA) with CRISPR/Cas12a holds great promise for point-of-care assay of bacterial infections, but still confronts challenges such as the input of unstable RNA, dependence on PAM, and multi-step operations. To address these challenges, we here present a tagged primer-based one-pot CRISPR/Cas12a assay, called TOP-CRISPR. Strategically, TOP-CRISPR utilized target gene-induced amplicons to generate crRNA sequences with the help of T7 RNA polymerase, thus eliminating the need for additional crRNA. The resulting Cas12a/crRNA complex were activated by the tag sequences at the end of specific primers rather than by the target amplicons, avoiding dependence on the PAM and disruption of the target amplicon by CRISPR/Cas12a. The ingenious design of tagged primer enables perfect compatibility between RPA and CRISPR/Cas12a system in a closed single tube, which is expected to overcome aerosol contamination caused by multi-step and open-cap operations. Under optimal conditions, the method was able to detect target bacteria down to ∼1 CFU/mL and the entire process took only less than 50 min, which is significantly better than the traditional two-step or one-step methods. By grafting the tag sequence onto different specific primers, bacteria such as Staphylococcus aureus, Escherichia coli O157:H7 and Pseudomonas aeruginosa has been detected, respectively. The practicability and robustness of the method was further validated by real samples. We hope that the method can be developed into a point-of-care detection tool for bacterial infections.