Rewriting regulatory DNA to dissect and reprogram gene expression.

Area of Science:

• Genomics
• Molecular Biology
• Gene Regulation

Background:

• Regulatory DNA sequences control cell-type-specific gene expression through transcription factor binding.
• Predicting the functional impact and programmability of regulatory DNA remains a significant challenge in molecular biology.

Purpose of the Study:

• To develop a high-throughput method for dissecting the function of endogenous regulatory DNA elements.
• To systematically reprogram regulatory elements and quantify the effects of designed edits on gene expression.

Main Methods:

• Development and application of Variant-EFFECTS (variant effects from flow-sorting experiments with CRISPR targeting screens).
• Introduction of hundreds of designed edits to endogenous regulatory DNA in specific genes and cell types.
• Quantification of gene expression changes using flow sorting and CRISPR screens.

Main Results:

• Dissection and reprogramming of three regulatory elements across two genes and two cell types.
• Identification of genomic context-specific effects of endogenous binding sites.
• Revealed cell-type-specific activities of transcription factor motifs and limitations of current computational prediction models.
• Demonstrated that small edits can tune gene expression over a wide dynamic range.

Conclusions:

• Variant-EFFECTS is a generalizable tool for dissecting regulatory DNA function.
• Identified genome editing strategies to precisely tune gene expression in an endogenous context.
• Findings suggest potential for prime-editing-based therapeutics targeting regulatory DNA for precise gene expression control.