Single-cell characterization of CRISPR-modified transcript isoforms with nanopore sequencing

Genome Biol. 2021 Dec 6;22(1):331. doi: 10.1186/s13059-021-02554-1.

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.

Keywords: Alternative splicing; Single-cell CRISPR screen; Single-cell long-read CRISPR screen; Single-cell long-read sequencing; Single-cell sequencing; Transcript isoform.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alternative Splicing
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Exons
  • Genomics
  • HEK293 Cells
  • Humans
  • Nanopore Sequencing / methods*
  • Neoplasm Proteins
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism*
  • RNA Isoforms / genetics
  • RNA Isoforms / metabolism
  • RNA Splicing
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Receptors for Activated C Kinase
  • Transcriptome

Substances

  • Neoplasm Proteins
  • Protein Isoforms
  • RACK1 protein, human
  • RNA Isoforms
  • RNA, Guide, CRISPR-Cas Systems
  • Receptors for Activated C Kinase