Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox

Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we develop and methodically test an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions. In parallel, we provide computational tools for experimental design and analysis. In this study, we demonstrate that junction-targeting achieves robust and isoform-specific RNA knockdown across diverse alternative isoform events, genes, and cell types. The majority of human genes can produce multiple isoforms, but studying their functional relevance requires tools to target specific isoforms. Here, the authors develop a CRISPR-based exon-exon junction-targeting strategy to achieve isoform-specific RNA knockdown.