CRYPTID-exon: streamlined detection of cryptic exons from RNA-seq data

Cryptic splicing has emerged as a pervasive feature of mammlian gene expression with recent studies discovering thousands of previously unannotated splice sites. Despite its prevalence, the functional consequences of this hidden layer of splicing remain largely unknown due to challenges in identifying the exact exonic regions introduced into mRNA transcripts. Here, we introduce a novel computational approach, CRYPTID-exon, that accurately predicts exon boundaries by modeling RNA-seq read coverage anchored on empirically derived splice sites. We use CRYPTID-exon to identify and characterize thousands of cryptic exons in nascent and mature RNA from human cells. Additionally, we demonstrate that CRYPTID-exon is well powered to identify exons that are sensitive to translation-mediated degradation processes. Finally, given the growing interest in leveraging cryptic exons to modulate gene expression levels, we use our approach to identify cryptic exons in disease-relevant genes. We see that targeting these exons with splice-switching antisense oligonucleotides (ASOs) can alter gene expression and splicing patterns of the parent genes. Our study provides a framework to systematically identify and characterize cryptic exons, which will enable downstream insights into their impact on mRNA stability and translation. Overall design: Targeted RNA sequencing: RNA-seq following combined transcript pulldown against 13 genes from nuclear RNA isolated from KNS60 cells. (Methods: Targeted sequencing of nuclear RNA from specific genes) Amplicon sequencing: PCR amplification using primer pairs covering: (a) upstream - exon region, (b) exon- downstream region, and (c) upstream - downstream of exon region, using cDNA generated from total RNA isolated from KNS60 cells. (Methods: Amplicon sequencing to validate cryptic exons) RNA-seq following ASO treatment: ASO treatment of KNS60 cells against target gene, using scrambled ASO (non-targeting control) and unstransfected (untreated) cells as controls, followed by transcript pulldown for target gene, and RNA-seq. (Methods:Antisense oligonucleotide (ASO) treatments)