Molina Vargas, A. M. | bioRxiv Science

bioRxiv2023

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 POINTSO_LICertain positions in the Cas13a crRNA-target-RNA duplex are particularly sensitive to mismatches. C_LIO_LIUnderstanding Cas13as allosteric activation pathway allowed us to develop novel high-fidelity Cas13a variants. C_LIO_LIThese Cas13a variants and crRNA design strategies achieve superior discrimination of SARS-CoV-2 strains. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/550755v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a3fd63org.highwire.dtl.DTLVardef@1509a82org.highwire.dtl.DTLVardef@cfcd7aorg.highwire.dtl.DTLVardef@bb2f0f_HPS_FORMAT_FIGEXP M_FIG New strategies to improve Cas13a RNA-detection specificity developed via mismatch tolerance profiling, uncovering features that modulate specificity, and structure-guided engineering of LbuCas13a. C_FIG

molecular biology↗

bioRxiv2023

RNA-mediated Allosteric Activation in CRISPR-Cas13a

Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. The protein uses a CRISPR RNA (crRNA) to target RNA sequences, which are cleaved by a composite active site formed by two Higher Eukaryotes and Prokaryotes Nucleotide (HEPN) catalytic domains. In this system, an intriguing form of allosteric communication controls RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory and RNA cleavage assays to decipher this activation mechanism. We show that the binding of a target RNA acts as an allosteric effector of the spatially distant HEPN catalytic cleft, by amplifying the allosteric signals over the dynamical noise, that passes through the buried HEPN interface. Critical residues in this region - N378, R973, and R377 - rearrange their interactions upon target RNA binding, and alter allosteric signalling. Alanine mutation of these residues is experimentally shown to select target RNA over an extended complementary sequence beyond guide-target duplex, for RNA cleavage. Altogether, our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of more selective RNA-based cleavage and detection tools.

biophysics↗