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 Isoform-specific Direct Capture CRISPR Perturb-seq9, a single cell screen to investigate the role of alternative promoters in driving cellular phenotypes. This approach enables the dual capture and sequencing of sgRNAs targeting individual promoters and evaluates the impact on their associated single-cell transcriptomes. We demonstrate that this approach leads to promoter-specific knockdown. In 13% of targeted promoters this induced an isoform-specific changes in the transcriptome demonstrating individual promoters can drive unique gene expression programs. We identify promoter-specific effects on cell cycle regulation for estrogen receptor 1 (ESR1) and validate these effects on proliferation. Together, this suggests that alternative promoter usage is a major regulator of transcriptomic complexit