New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity.

The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics. KEY POINTS ![Figure][1] New strategies to improve Cas13a RNA-detection specificity developed via mismatch tolerance profiling, uncovering features that modulate specificity, and structure-guided engineering of LbuCas13a. ### Competing Interest Statement M.R.O is an inventor on patent applications related to CRISPR-Cas systems and uses thereof. M.R.O is a member of the scientific advisory boards for Dahlia Biosciences and Locana Bio, and an equity holder in Dahlia Biosciences and LocanaBio. A.M.M.V., S.S, P.R.A., A.P, G.P. and M.R.O are co-inventors on patent applications filed by the University of Rochester and University of California, Riverside relating to work in this manuscript. [1]: pending:yes