PXD060902

PXD060902 is an original dataset announced via ProteomeXchange.

Title	Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox
Description	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 developed and methodically tested an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions in the mature RNA. We performed a high-throughput essentiality screen, quantitative RT-PCR assays, and PacBio long read sequencing to affirm our ability to specifically target and robustly knockdown individual RNA isoforms. Using the example gene RBFOX2, we validated protein-level changes and assessed the functional impact of isoform-specific knockdown. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions annotated in GENCODE for multi-isoform genes and our gRNA efficacy predictions, we estimate that our junction-centric strategy can uniquely target up to 89% of human RNA isoforms, including 50,066 protein-coding and 11,415 lncRNA isoforms. Importantly, this specificity spans all transcriptional and splicing events, including exon skipping and inclusion, alternative 5’ and 3’ splice sites, and alternative starts and ends.
HostingRepository	PanoramaPublic
AnnounceDate	2025-06-25
AnnouncementXML	Submission_2025-06-25_13:29:40.616.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Erin Jeffery
SpeciesList	scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2025-02-17 21:22:47	ID requested
⏵ 1	2025-06-25 13:29:41	announced

no publication

submitter keyword: RBFOX2, HUES66, knockdown, PRM, tMS2

Gloria Sheynkman
contact affiliation	University of Virginia School of Medicine
contact email	gs9yr@virginia.edu
lab head
Erin Jeffery
contact affiliation	University of Virginia Dept of Molecular Biology and Biological Physics
contact email	edf4n@virginia.edu
dataset submitter

Panorama Public dataset URI