The facile and economical identification of pathogenic bacteria, especially their antibiotic-resistance, is crucial in the realm of human health and safety. The presence of Escherichia coli (E. coli) is considered as an indicator of water contamination and is closely related to human health. Herein, inspired by the biocatalysis of bacterial surfaces, we developed a simple and cost-effective colorimetric- and electrochemical-based bioassay that is capable of analyzing both the presence of E. coli and its relative level of antibiotic resistance. In this approach, p-benzoquinone is used as a redox mediator to monitor the bacterial concentration and specifically distinguish E. coli from four other common clinical bacteria, namely, Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), Salmonella pullorum (S. pullorum), and Streptococcus mutans (S. mutans). A visible color change, captured with a smartphone using a “light box”, without relying on any complex instruments, can reflect the concentration of bacteria. The accurate quantification of E. coli was investigated with an electrochemical system in the concentration ranges of 1.0 × 103 to 1.0 × 109 CFU/mL. We further demonstrated the capability of the presented biosensor in identifying drug-resistant bacteria with two artificially induced antibiotic-resistant bacteria. Therefore, the presented bioassay is not only capable of detecting E. coli with high sensitivity and specificity but also provides a rapid solution to evaluate E. coli antibiotic resistance.

The Cas13a system has great potential in RNA interference and molecular diagnostic fields. However, lacking guidelines for crRNA design hinders practical applications of the Cas13a system in RNA editing and single nucleotide polymorphism identification. This study posits that crRNAs with hairpin spacers improve the specificity of CRISPR/Cas13a system (termed hs‐CRISPR). Gibbs free energy analysis suggests that the hairpin‐spacer crRNAs (hs‐crRNAs) suppress Cas13a's affinity to off‐target RNA. A hepatitis B virus DNA genotyping platform is established to further validate the high‐specificity of hs‐CRISPR/Cas13a system. Compared to ordinary crRNA, hs‐crRNAs increase the specificity by threefold without sacrificing the sensitivity of the CRISPR/Cas13a system. Furthermore, the mechanism of the Cas13a/hs‐crRNA/target RNA composition is elucidated with theoretical simulations. This work builds on the fundamental understanding of Cas13a activation and offers significant improvements for the rational design of crRNA for the CRISPR/Cas13a system.