AltEx-BE: a tool for designing splice-site-targeting gRNAs for base editing-mediated exon skipping with exon-level...

Pre-mRNA splicing is a conserved post-transcriptional regulatory process in eukaryotes, and mis-splicing is associated with numerous diseases. Therefore, experimentally induced exon skipping provides an important strategy for exon-level analysis of gene function. Exons can be broadly classified by their usage patterns across transcripts: some are shared by all transcripts, whereas others are selectively used across transcripts. Skipping the former affects all transcripts and often results in gene-level effects, while skipping the latter reflects functional diversity across transcripts. Therefore, considering characteristics of target exons is important to enhance interpretability of exon-level functional analysis. Recent advances in base editing enable exon skipping by introducing point mutations at splice sites, providing an approach for exon-level analysis. Accordingly, several guide RNA (gRNA) design tools have been developed for the purpose of inducing exon skipping. However, existing tools do not provide exon-level properties, including exon usage patterns, reading frame, and coding status. As a result, selecting target exons appropriate for specific research objectives remains difficult. Here we developed AltEx-BE, an automated gRNA design tool for base editing-mediated splice site disruption. AltEx-BE uses transcript annotations and reference genome sequences as inputs to classify each exon by usage pattern and to generate gRNA candidates compatible with several base editing tools. The tool also provides annotations, including coding status, to facilitate interpretation of outcomes of exon skipping. The tool promotes base editing-mediated exon skipping experiments across model organisms and enhances the interpretability of experimental outcomes.