CRISPR/Cas12a‐Enabled Multiplex Biosensing Strategy Via an Affordable and Visual Nylon Membrane Readout

Most multiplex nucleic acids detection methods require numerous reagents and high‐priced instruments. The emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas has been regarded as a promising point‐of‐care (POC) strategy for nucleic acids detection. However, how to achieve CRISPR/Cas multiplex biosensing remains a challenge. Here, an affordable means termed CRISPR‐RDB (CRISPR‐based reverse dot blot) for multiplex target detection in parallel, which possesses the advantages of high sensitivity and specificity, cost‐effectiveness, instrument‐free, ease to use, and visualization is reported. CRISPR‐RDB integrates the trans‐cleavage activity of CRISPR‐Cas12a with a commercial RDB technique. It utilizes different Cas12a‐crRNA complexes to separately identify multiple targets in one sample and converts targeted information into colorimetric signals on a piece of accessible nylon membrane that attaches corresponding specific‐oligonucleotide probes. It has demonstrated that the versatility of CRISPR‐RDB by constructing a four‐channel system to simultaneously detect influenza A, influenza B, respiratory syncytial virus, and SARS‐CoV‐2. With a simple modification of crRNAs, the CRISPR‐RDB can be modified to detect human papillomavirus, saving two‐thirds of the time compared to a commercial PCR‐RDB kit. Further, a user‐friendly microchip system for convenient use, as well as a smartphone app for signal interpretation, is engineered. CRISPR‐RDB represents a desirable option for multiplexed biosensing and on‐site diagnosis.