High-resolution RNA knockdown profiles of small RNA and Cas13 systems

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ABSTRACT Programmable RNA-targeting systems, including RNA interference (RNAi) and CRISPR-Cas13, are transformative tools for research and therapeutics. However, a lack of high-resolution comparative data obscures whether these mechanistically distinct platforms share fundamental rules for target-site selection. To address this, we developed shRNAone, an engineered scaffold that eliminates processing heterogeneity to guarantee guide biogenesis at single-nucleotide precision. Deploying this platform to systematically decouple microRNA pairing rules in cellulo , we demonstrate that while seed pairing drives primary target recognition, 3′-supplementary pairing is strictly essential for specific transcript repression. Furthermore, single-nucleotide shRNAone tiling screens accurately predict siRNA efficacy and reveal clustered, highly permissive silencing hotspots localized within 3′ UTRs. By mapping CasRx and PspCas13b silencing landscapes across the identical mRNA, we show that these hotspots represent intrinsic structural vulnerabilities governed by local RNA accessibility. However, while shRNAone and CasRx activity is largely restricted to the 3′ UTR, PspCas13b efficiently targets the coding sequence, highlighting its unique capacity to overcome ribosome occupancy. Together, these high-resolution profiles define the shared and distinct structural barriers governing diverse RNA-targeting systems and provide a rationally designed, transcriptome-wide guide resource for PspCas13b applications.