This technology enables high-throughput, single-cell screening of gene regulatory sequences using barcoded viral vectors and sequencing, allowing precise identification and quantification of active regulatory elements in specific cell types for gene therapy, disease research, and synthetic biology.
Background
The field of gene regulation is fundamental to understanding how genes are selectively activated or silenced in different cell types, developmental stages, or disease states. Regulatory gene elements (RGEs), such as promoters and enhancers, dictate the spatial and temporal patterns of gene expression, and their precise identification is crucial for applications in gene therapy, functional genomics, and synthetic biology. In gene therapy, for example, the ability to target gene expression to specific cell types can greatly enhance treatment efficacy while minimizing off-target effects. Similarly, in basic research, mapping the activity of RGEs at single-cell resolution is essential for unraveling the complexities of cellular identity and function within heterogeneous tissues. The growing demand for personalized medicine and targeted genetic interventions underscores the need for scalable, high-throughput methods to discover and characterize RGEs with high specificity and quantitative accuracy. Current approaches to RGE discovery and validation are limited by several significant challenges. Traditional methods often rely on bulk assays or reporter constructs tested one at a time, which are labor-intensive, low-throughput, and provide only limited information about cell-type specificity. These approaches typically cannot resolve RGE activity at the single-cell level, making it difficult to accurately map regulatory sequences to the diverse cell types present in complex tissues. Moreover, existing multiplexed techniques may lack the quantitative rigor or the ability to directly link RGE activity to individual cells, particularly in vivo. As a result, researchers face substantial barriers in efficiently screening large libraries of candidate RGEs, assessing their functional strength, and ensuring that they drive expression only in desired cell populations. These limitations hinder the pace of discovery and the development of precise, cell-targeted genetic therapies.
Technology Description
This technology is a high-throughput, single-cell resolution platform designed to screen and characterize gene regulatory sequences (RGEs), such as promoters and enhancers, within complex biological tissues. Central to the system is an adeno-associated virus (AAV) vector—though compatible with other delivery methods—carrying two expression cassettes: one universally marking all infected cells and another for RGE screening. The RGE screening cassette features an insertion site for candidate regulatory sequences, a minimal promoter, a unique barcode for each RGE, an internal polyA domain, a reporter gene, and a 3’ polyadenylation signal. Upon delivery to target cells, the system captures reporter transcripts using poly-thymidine beads, tagging each with a cell-specific barcode and a unique molecular identifier (UMI) during cDNA synthesis. High-throughput sequencing and computational analysis then match RGE barcodes to cell barcodes, enabling precise identification of active RGEs in specific cell types and quantifying their transcriptional activity. What differentiates this technology is its ability to multiplex hundreds of candidate RGEs in a single experiment, directly within living tissues, and resolve their activity at the level of individual cells. The dual polyA strategy in the reporter transcript enhances capture efficiency and reduces sequencing artifacts, ensuring robust and accurate data. Unlike traditional methods that are labor-intensive and limited to one-at-a-time testing, this platform enables scalable, quantitative, and cell-type-specific screening, which is invaluable for gene therapy, functional genomics, and synthetic biology. Its versatility across delivery methods, cell types, and species—along with the integration of single-cell transcriptomics—makes it uniquely positioned to accelerate the discovery of regulatory elements for targeted genetic interventions and personalized medicine.
Benefits
- Enables high-throughput, multiplexed screening of gene regulatory sequences (RGEs) at single-cell resolution.
- Allows precise identification of cell type-specific promoter and enhancer activity within complex tissues.
- Quantifies transcriptional strength of RGEs using unique molecular identifiers (UMIs) for accurate measurement.
- Facilitates discovery of RGEs that drive targeted gene expression, improving safety and efficacy in gene therapy.
- Compatible with various delivery methods including viral vectors and non-viral approaches, adaptable to in vitro and in vivo studies.
- Supports simultaneous analysis of endogenous transcripts to confirm cell identity and refine regulatory mapping.
- Accelerates functional genomics, synthetic biology, and disease modeling by linking regulatory sequences to cellular function.
- Incorporates a universal infection marker to identify all infected cells, ensuring relevant data collection.
Commercial Applications
- Cell type-specific gene therapy design
- Multiplexed promoter/enhancer screening
- Single-cell functional genomics profiling
- Precision disease model development
- Synthetic promoter validation
This high-throughput platform screens gene regulatory sequences (RGEs) at single-cell resolution. It employs a viral vector delivering barcoded RGEs linked to a reporter. Transcripts, containing RGE, cell, and unique molecular identifiers, are captured and sequenced. Computational analysis quantifies RGE activity in specific cell types, enabling multiplexed functional genomics and gene therapy applications.
Intellectual Property
Provisional patent filed 08/28/2025
