Abstract
We developed a single-cell approach to detect CRISPR-modified mRNA transcript structures. This method assesses how genetic variants at splicing sites and splicing factors contribute to alternative mRNA isoforms. We determine how alternative splicing is regulated by editing target exon-intron segments or splicing factors by CRISPR-Cas9 and their consequences on transcriptome profile. Our method combines long-read sequencing to characterize the transcript structure and short-read sequencing to match the single-cell gene expression profiles and gRNA sequence and therefore provides targeted genomic edits and transcript isoform structure detection at single-cell resolution.
Highlights
For any gene’s messenger RNA, alternative splicing events lead to a diverse set of transcripts that have different combinations of exons
This method assesses how genetic variants at splicing sites and splicing factors contribute to alternative messenger RNA (mRNA) isoforms
We found that CELF2 targeted cells had a higher percentage of full-length myosin light chain 6 (MYL6) transcript isoforms compared to cells targeted by other splicing factors (1.44-fold, P = 1.9e−14, Additional file 1: Fig S14B), indicating that disrupting CELF2 reduced the occurrence of exon-skipping
Summary
For any gene’s messenger RNA (mRNA), alternative splicing events lead to a diverse set of transcripts that have different combinations of exons. Referred to as transcript isoforms, these different mRNA species result from genetic variants at intronexon junctions and the functional contribution of different splicing factors. This diversity of transcripts provides cells with an added level of transcriptional regulation. Most studies examining the structure and function of isoform variation have relied on CRISPR or other genome engineering methods. These experiments involve introducing a single genetic variant within an exon/intron junction or knocking out a splicing factor. Conventional short-read approaches may not resolve important transcript isoform features that are present in only a subset of cells since these require complex bioinformatic
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