STARRseQ

STARR-seq stands for Self-Transcribing Active Regulatory Region sequencing. It is a high-throughput assay used to identify and quantify regulatory DNA elements, especially enhancers, by measuring their ability to activate transcription in a cellular context. It enables genome-wide mapping of active regulatory regions and allows comparison across cell types or conditions.

Core method: A library of DNA fragments covering candidate regulatory regions is cloned into a plasmid so that each fragment is placed within the transcript of a reporter gene, downstream of a minimal promoter. If a fragment has enhancer activity, it increases transcription of the reporter RNA, which includes the fragment sequence. The plasmid library is transfected or delivered into cells; after expression, total RNA is extracted and the reporter transcripts are sequenced. The abundance of each fragment in RNA (active signal) is compared to its abundance in the input DNA library to assign an activity score, often using RNA/DNA ratios or log fold change.

Applications: STARR-seq is used to identify enhancers in humans and model organisms, validate predicted regulatory elements, and characterize tissue- or condition-specific activity. It also enables assessment of how genetic variants may influence regulatory function. By providing genome-wide enhancer activity maps, STARR-seq supports studies of gene regulation and the regulatory architecture underlying complex traits.

Variants and limitations: The assay relies on a plasmid-based context that may not capture all native chromatin features, and results can be influenced by fragment length and promoter choice. Delivery efficiency and construct design can introduce biases. Adaptations exist, such as lentiviral STARR-seq, to enable delivery to difficult cell types or tissues and to explore regulatory activity in different cellular contexts.