Integrative dissection of gene regulatory elements at base resolution

Abstract

Although vast numbers of putative gene regulatory elements have been cataloged, the sequence motifs and individual bases that underlie their functions remain largely unknown. Here we combine deep learning, epigenetic perturbations and base editing to dissect regulatory sequences within the exemplar immune locus encoding CD69. Focusing on a differentially accessible and acetylated upstream enhancer, we find that the complementary strategies converge on a ~150 base interval as critical for CD69 induction in stimulated Jurkat T cells. We pinpoint individual cytosine to thymine base edits that markedly reduce element accessibility and acetylation, with corresponding reduction of CD69 expression. The most potent base edits may be explained by their effect on binding competition between the transcriptional activator GATA3 and the repressor BHLHE40. Systematic analysis of GATA and bHLH/Ebox motifs suggests that interplay between these factors plays a general role in rapid T cell transcriptional responses. Our study provides a framework for parsing gene regulatory elements in their endogenous chromatin contexts and identifying operative engineered variants.

Rewriting regulatory DNA to dissect and reprogram gene expression

“…Massively parallel reporter assays have revealed how transcription factor binding sites combine to drive enhancer and promoter activity in plasmids [15][16][17][18][19][20][21][22] , but do not properly model key aspects of genomic context. CRISPR nucleases and base editing have been coupled with single-cell or sorting-based readouts to identify and dissect regulatory elements 5,[23][24][25][26][27][28][29][30][31] , but lack the flexibility to precisely reprogram regulatory sequences, depend on fortuitous positioning of editing sites to disrupt or introduce transcription factor binding sites, and often produce multiple possible edits per gRNA that lead to challenges in data interpretation 32 . The development of CRISPR prime editing, which provides the ability to precisely delete or insert designed sequences up to dozens of base pairs 33 , promises to address some of these challenges.…”

Enhancer regulatory networks globally connect non-coding breast cancer loci to cancer genes

“…Likewise, endogenous CRISPR-mediated saturation mutagenesis of the BCL11A enhancer identified the key base positions for proper regulation of fetal hemoglobin 66,119 . Recently developed technologies including prime editing and base editing will also play important roles to address this challenge 120,121 . Ultimately, interpreting non-coding variants will require orthogonal approaches to experimentally link enhancers and their variants to molecular and cellular phenotypes relevant to cancer.…”

Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes

“…Resolving the individual bases and variants that underlie the activity of enhancers remains challenging as these and other genomic regulatory elements must ideally be examined in their native chromatin context. Although we and others have used CRISPR base editors to characterize enhancers [38][39][40] , such approaches require separate sgRNAs for each narrow target window and may be limited by the occurrence of PAM sites (only ~30% of locations are targetable with NGG PAM sites). Furthermore, artificial linkage between variants due to correlated mutations in an editing window limits the ability of base editors to resolve the functions of single bases 41 .…”

“…Although we and others have used CRISPR base editors to characterize enhancers 38–40 , such approaches require separate sgRNAs for each narrow target window and may be limited by the occurrence of PAM sites (only ∼30% of locations are targetable with NGG PAM sites). Furthermore, artificial linkage between variants due to correlated mutations in an editing window limits the ability of base editors to resolve the functions of single bases 41 .…”