Recruitment of transcriptional effectors by Cas9 creates cis-regulatory elements and demonstrates distance-dependent transcriptional regulation.

Regulation of cardiac gene expression, especially for sarcomeric genes, is crucial for heart function and is often mediated by distal regulatory elements such as enhancers and repressors. Despite their importance, the link between transcription factor recruitment to distant genomic regions and their ability to modulate gene expression remains unclear. Here, we used dead Cas9 to target either viral or endogenous cardiac transcription factor domains to naive genomic sites lacking chromatin accessibility or active regulatory marks. Remarkably, these sites underwent epigenetic remodeling, altering both local and distal promoter chromatin and significantly changing gene expression, even across insulating loci. The degree of transcriptional activation or repression varied non-linearly with the distance between the regulatory site and the gene. These findings broaden the traditional framework of enhancer and repressor function, revealing that virtually any DNA site can become regulatory, and provide fundamental insights into the rules governing gene expression in the heart.