@article {Kim2021.07.01.450805, author = {Heon Seok Kim and Susan M Grimes and Anna C Hooker and Billy T Lau and Hanlee P Ji}, title = {Single cell characterization of CRISPR-modified transcript isoforms with nanopore sequencing}, elocation-id = {2021.07.01.450805}, year = {2021}, doi = {10.1101/2021.07.01.450805}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Transcript isoforms are mRNAs that arise from alternative splicing events. During RNA processing, different combinations of a gene{\textquoteright}s exons lead to a diverse set of isoforms. Polymorphisms or mutations at splice junctions can generate alternative splicing events. Various splicing factors also impact the representation of a gene{\textquoteright}s transcript isoforms. To assess how these two features contribute to alternative splicing, we developed a single cell approach to introduce CRISPR edits that modify mRNA transcript structure. Our method combines (1) long-read sequencing to characterize the expressed transcripts and identify the edit at single cell resolution; (2) short-read sequencing to match the single cell gene expression profiles of the cells with the altered isoform. First, we modify target exon-intron segments with CRISPR-Cas9. Second, using cDNAs with cell barcodes, we use long read sequencing to directly identify the changes in transcript isoforms from the targeted CRISPR edits. As a variation on this approach, we also determined how modifying specific splicing factors influence isoform expression and structure. Overall, we demonstrate how the integration of single cell long read analysis and CRISPR engineering can be used to directly confirm transcript isoform and target genomic edits at single cell resolution. This approach will improve our understanding of the role of alternative splicing in transcriptional regulation.Competing Interest StatementThe authors have declared no competing interest.}, URL = {https://www.biorxiv.org/content/early/2021/07/02/2021.07.01.450805}, eprint = {https://www.biorxiv.org/content/early/2021/07/02/2021.07.01.450805.full.pdf}, journal = {bioRxiv} }