Rewriting regulatory DNA to dissect and reprogram gene expression.

Area of Science:

• Genomics
• Molecular Biology
• Systems Biology

Background:

• Regulatory DNA elements like enhancers and promoters control cell-specific gene expression through transcription factor binding.
• Predicting and mapping the precise regulatory effects of DNA sequences is challenging due to a lack of scalable editing and quantification methods.
• Understanding these regulatory mechanisms is crucial for deciphering gene expression patterns and disease pathologies.

Purpose of the Study:

• To develop and validate a scalable method for precisely editing regulatory DNA and quantifying its effects on gene expression.
• To investigate the impact of designed DNA sequence variants on the expression of the PPIF gene in immune cells.
• To benchmark and improve computational models of gene regulation using experimental data.

Main Methods:

• Developed Variant-FlowFISH, combining pooled CRISPR prime editing with RNA fluorescence in situ hybridization and cell sorting.
• Applied Variant-FlowFISH to mutagenize and rewrite regulatory sequences in the enhancer and promoter of the PPIF gene in two immune cell lines.
• Designed and tested hundreds of DNA sequence variants to map their quantitative effects on gene expression.

Main Results:

• Identified 497 out of 672 variant-cell type pairs that significantly affect PPIF gene expression.
• Observed that single transcription factor binding site disruptions can cause substantial expression changes (up to -50%).
• Demonstrated cell type-specific variant effects and found that existing predictive models struggle with certain variant types.

Conclusions:

• Variant-FlowFISH is a powerful tool for mapping regulatory DNA variant effects and transcription factor binding sites.
• The study provides valuable data for improving computational models of gene regulation.
• The method enables the rational design and reprogramming of regulatory DNA sequences for specific gene expression outcomes.