Integrative dissection of gene regulatory elements at base resolution

Chen, Zeyu; Javed, Nauman; Moore, Molly M.; Wu, Jingyi; Sun, Gary; Vinyard, Michael; Collins, Alejandro; Pinello, Luca; Najm, Fadi J.; Bernstein, B

doi:10.1016/j.xgen.2023.100318

Cited by 8 publications

(4 citation statements)

References 67 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Wang,

Armendariz,

Wang

et al. 2023

Preprint

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Genetic studies have associated thousands of enhancers with breast cancer. However, the vast majority have not been functionally characterized. Thus, it remains unclear how variant-associated enhancers contribute to cancer. Here, we perform single-cell CRISPRi screens of 3,512 regulatory elements associated with breast cancer to measure the impact of these regions on transcriptional phenotypes. Analysis of >500,000 single-cell transcriptomes in two breast cancer cell lines shows that perturbation of variant-associated enhancers disrupts breast cancer gene programs. We observe variant-associated enhancers that directly or indirectly regulate the expression of cancer genes. We also find one-to-multiple and multiple-to-one network motifs where enhancers indirectly regulate cancer genes. Notably, multiple variant-associated enhancers indirectly regulate TP53. Comparative studies illustrate sub-type specific functions between enhancers in ER+ and ER- cells. Finally, we developed the pySpade package to facilitate analysis of single-cell enhancer screens. Overall, we demonstrate that enhancers form regulatory networks that link cancer genes in the genome, providing a more comprehensive understanding of the contribution of enhancers to breast cancer development.

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Section: Discussionmentioning

confidence: 99%

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

Wang,

Armendariz,

Wang

et al. 2023

Preprint

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“…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 .…”

Section: Mutagenesis Of Noncoding Regulatory Elements Uncovers Functi...mentioning

confidence: 99%

“…6B). Our base editing tiling screen previously identified this artificial variant, which disrupts a GATA transcription factor motif 41 . We also validated G>A transitions at both bases 4879 and 4880 that suppress CD69 induction (Supplementary Fig.…”

Section: Mutagenesis Of Noncoding Regulatory Elements Uncovers Functi...mentioning

confidence: 99%

Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes

Chen,

Wythes

et al. 2024

Preprint

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A major challenge in human genomics is to decipher the context specific relationship of sequence to function. However, existing tools for locus specific hypermutation and evolution in the native genome context are limited. Here we present a novel programmable platform for long-range, locus-specific hypermutation called helicase-assisted continuous editing (HACE). HACE leverages CRISPR-Cas9 to target a processive helicase-deaminase fusion that incurs mutations across large (>1000 bp) genomic intervals. We applied HACE to identify mutations in MEK1 that confer kinase inhibitor resistance, to dissect the impact of individual variants in SF3B1-dependent mis-splicing, and to evaluate noncoding variants in a stimulation-dependent immune enhancer of CD69. HACE provides a powerful tool for investigating coding and noncoding variants, uncovering combinatorial sequence-to-function relationships, and evolving new biological functions.One Sentence SummaryWe developed a tool for continuous, long-range, targeted diversification of endogenous mammalian genomes and used it to explore the function of genetic variants in both coding and non-coding regions.

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“…Importantly, the authors also performed pooled single-cell genotyping to link the nucleotide variants to the phenotype. In a separate study, Chen et al, took a tiered approach by combining deep learning-based methodologies with CRISPRi and base editing screens to identify variants in enhancers of CD69 ( Chen et al, 2023 ). Finally, Morris et al, extended this base editing screening strategy to functionally test GWAS variants for blood traits with a single-cell RNA-Seq readout ( Morris et al, 2023 ).…”

Section: Introductionmentioning

confidence: 99%

Breaking enhancers to gain insights into developmental defects

Armendariz

Sundarrajan

Hon

2023

eLife

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Despite ground-breaking genetic studies that have identified thousands of risk variants for developmental diseases, how these variants lead to molecular and cellular phenotypes remains a gap in knowledge. Many of these variants are non-coding and occur at enhancers, which orchestrate key regulatory programs during development. The prevailing paradigm is that non-coding variants alter the activity of enhancers, impacting gene expression programs, and ultimately contributing to disease risk. A key obstacle to progress is the systematic functional characterization of non-coding variants at scale, especially since enhancer activity is highly specific to cell type and developmental stage. Here, we review the foundational studies of enhancers in developmental disease and current genomic approaches to functionally characterize developmental enhancers and their variants at scale. In the coming decade, we anticipate systematic enhancer perturbation studies to link non-coding variants to molecular mechanisms, changes in cell state, and disease phenotypes.

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Integrative dissection of gene regulatory elements at base resolution

Cited by 8 publications

References 67 publications

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

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

Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes

Breaking enhancers to gain insights into developmental defects

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