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

Megan D. Schertzer (), Andrew Stirn, Keren Isaev, Laura Pereira, Stella H. Park, Anjali Das, Aline Réal, Erin D. Jeffery, Claire Harbison, Hans-Hermann Wessels, Gloria M. Sheynkman, Neville E. Sanjana and David A. Knowles ()
Additional contact information
Megan D. Schertzer: New York Genome Center
Andrew Stirn: New York Genome Center
Keren Isaev: New York Genome Center
Laura Pereira: New York Genome Center
Stella H. Park: New York Genome Center
Anjali Das: New York Genome Center
Aline Réal: New York Genome Center
Erin D. Jeffery: University of Virginia
Claire Harbison: New York Genome Center
Hans-Hermann Wessels: New York Genome Center
Gloria M. Sheynkman: University of Virginia
Neville E. Sanjana: New York Genome Center
David A. Knowles: New York Genome Center

Nature Communications, 2025, vol. 16, issue 1, 1-19

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-62066-5

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