CRISPR-Cas13d is increasingly used for RNA knockdowns due to its programmability, but off-target RNA binding and cleavage of near-cognate RNAs hinder its broader adoption. Here, we explore the mechanisms of nuclease activation by solving seven ternary cryo-electron mi-croscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal a series of active, intermediate, and inactive states that illustrate a detailed activation mechanism. The crRNA undergoes dramatic conformational changes upon target RNA binding, with the helical-1 domain transitioning from an initially docked state with the N-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics reveal that a single proximal mismatch preserves nanomolar binding affinity but completely abolishes nuclease activity by trapping Cas13d in an inactive state. We identify an active site loop in the HEPN domains that regulates substrate accessibility, with alanine scanning mutagenesis revealing both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d’s exquisite mismatch surveillance and provide a mechanistic framework for engineering RNA-targeting specificity and activity across HEPN nuclease family members.
Chia-Wei Chou, Selma Sinan, Hung-Che Kuo et al.· bioRxiv· 0 citations
The modular structure and energetics of RNA simplifies its folding. Leveraging this modularity, we introduce Rho (ρ) analysis to systematically dissect RNA conformational pathways. ρ analysis uses isolated RNA secondary or tertiary contacts as external standards to provide insights not possible via the “internal” comparisons of traditional ϕ analysis. Equivalent effects of a mutation on the folding rate constant of the RNA of interest and the thermodynamic stability of the isolated contact indicate that the mutated interaction is fully formed prior to the rate-limiting transition state; the absence of a kinetic effect indicates that the interaction is formed after this transition state. Comparisons with properties of the isolated contact provide additional insights about conformational pathways. We demonstrate ρ analysis by dissecting Tetrahymena group I intron folding pathways, using a split intron in which the P5abc subdomain assembles with the intron core through three tertiary contacts. We uncover multiple folding pathways and modulation in pathway flux that are readily understood from the energetic properties of the constituent RNA motifs. Extending these concepts to RNA-guided DNA recognition by CRISPR-Cas12a, crRNA–DNA mismatches give substantial ϕ values across much of the target, indicating a late transition state in binding. Thermodynamic penalties from mismatches support modular base-pairing energetics and define an upper bound on DNA target specificity. Our results establish ρ analysis as a general framework to probe RNA conformational pathways and function. It is straightforward to implement and can be readily applied in vitro and in cells.
Brant Gracia, Sarah E Nielson, Daniel Herschlag et al.· bioRxiv· 0 citations