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Effect of promoter compatibility and sequence positioning on high-throughput identification of cell type-specific enhancers using STARR-CRAAVT in HepG2 and HaCaT cells

ABSTRACT: Tuning transgene expression through novel combinations of enhancers and promoters holds great potential for adeno-associated virus (AAV)-based gene therapies. Here, we present STARR-CRAAVT, a STARR-Seq-based platform to screen for enhancers in the AAV context. Employing two in-vitro models, HepG2 and HaCaT cells, we aimed to implement STARR-CRAAVT for the identification of cell type-specific enhancers. We integrated epigenetic datasets into an in-silico library of putative HepG2 enhancers and captured corresponding fragments from sheared genomic DNA. The fragments were processed into AAV libraries and applied to HepG2 and HaCaT cells. Comparative STARR-CRAAVT analysis revealed a selective activity of the libraries confirming our HepG2-directed in-silico design and the specificity of single enhancer/promoter combinations could be validated using luciferase reporter assays. In addition, we scrutinized the impact of key experimental parameters on enhancer identification and found that enhancer activity was significantly influenced by the used promoter. Furthermore, switching the enhancer location in reporter assays revealed that the level of enhancer activity is highly dependent on the position in the AAV genome. Taken together, this study demonstrates that STARR-CRAAVT can be employed to identify cell type-specific enhancers for AAV vectors and highlights promoter preference and enhancer positioning as key considerations for STARR-CRAAVT campaigns.

ORGANISM(S): Homo sapiens

PROVIDER: GSE298485 | GEO | 2026/03/19

REPOSITORIES: GEO

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