Systematic analysis, identification, and use of CRISPR/Cas13a–associated crRNAs for sensitive and specific detection of the lcrV gene of Yersinia pestis

•Development of a Cas13a-based assay to detect Yersinia pestis lcrV.•Demonstration of crRNA performance diversity in a Cas13a-based diagnostic test.•Description and results of a streamlined crRNA screening protocol.•Discussion of considerations to achieve rapid crRNA design for CRISPR diagnostics. CRISPR-associated proteins that produce a signal in the presence of a target nucleic acid represent potentially powerful tools for diagnostics, but they also exhibit shortfalls that plague many CRISPR systems. For instance, not all targets elicit robust activity, which challenges the timely development of sensitive assays, and though many such tests have been reported, they often avoid discussion of the crRNA design and screening process. Here, motivated by the desire to detect the Yersinia pestis lcrV virulence gene, we detail the process involved in developing components for a CRISPR-based test that provides sensitive and specific identification of this sequence using Cas13a. This includes detailing the diversity of crRNA performance, identifying sequence that enable detection with attomolar sensitivity and species-level specificity, and presenting a method for simple streamlining of the crRNA screening process to allow for the high-throughput testing required for developing assay design rules in the future.