Oral Presentation 50 Years Shine-Dalgarno Symposium 2023

Single-cell CRISPR Reveals the Role of Alternative Promoter Usage in Shaping the Transcriptome (#24)

Helen E King 1 2 , Helaine Graziele S Vieira 1 3 , Javier Fernandez 1 3 , Robert Weatheritt 1 3
  1. the Garvan Institute, Darlinghurst, New South Wales, Australia
  2. St. Vincent Clinical School, University of New South Wales, Darlinghurst, New South Wales, Australia
  3. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia

Alternative promoter usage generates multiple isoforms from a single gene locus. This is a major source of biological diversity and occurs in more than 50% of human protein-coding genes5. The consequences of forming these isoforms range from a change in translational efficiency1,2 to the generation of different protein domains3,4. Aberrant activation or silencing of alternative promoters is correlated with the severity and progression of cancer 6,7,8. However, there is a lack of high-throughput experiments to investigate their function on the transcriptome. Therefore, our functional understanding of the causative effect of alternative promoter regulation is limited.


Here, we present a single-cell Direct Capture CRISPR Perturb-seq9 screen to investigate the role of alternative promoters within transcription factors and chromatin factors. Our application of this 10x technology facilitates the dual capture and sequencing of expressed sgRNAs with their associated single-cell transcriptomes. We first identified sites of alternative promoter usage across the MCF-7 cell line. Then we designed novel guides that caused a promoter-specific knockdown. By harnessing the genotype-phenotype relationship, we observed isoform-specific expression. In regulating promoter usage, we found an isoform-specific expression pattern that regulates and diversifies distinct transcriptomes. Together, this suggests that alternative promoter usage is a major regulator of transcriptomic complexity.

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