Optimization and Modeling of Crrna Design for Pathogen Detection By Cas13 Enzymes

Type VI Cas proteins possess non-discriminatory endonuclease activity that is triggered by the binding of the CRISPR ribonucleoprotein complex to target sequences. This feature has been coopted to enable customizable detection of nucleic acids through coupling nuclease activity to molecular beacons, providing the potential to create made-to-order, point-of-care diagnostics with strain discrimination capabilities. However, the speed of development, the sensitivity, and the specificity of this assay is dependent upon, in part, features of the crRNA, as well as the Cas protein used. We are interested in improving protocols for crRNA development that minimizes cost and development time. Toward this goal we have synthesized and tested several crRNAs tiled against loci in Yersinia species, for use with Cas13a proteins from Leptotrichia buccalis and Leptotrichia wadei, and determined features (including GC content and predicted, ancillary secondary structure) of the crRNAs that provided the most robust, specific readout using fluorescent small RNA reporters. Our results indicate that false positives are a concern with Cas13a orthologs from certain species, and that signal intensity varies significantly between different but closely related crRNAs. However, certain conditions and crRNAs can provide discriminatory capabilities down to the level of 1 and 2 nucleotides, which is sufficient to distinguish many closely related bacterial strains.