Maitreya Das, Deepro Banerjee, Ayaan Hossain, Matthew Jensen, Saie Mogre et al.
Transcriptional regulation is mediated by enhancers, yet how genetic perturbations alter enhancer activity and gene expression remains poorly understood. We developed UDI-UMI-STARR-seq, which integrates dual indexes and unique molecular identifiers, and combined it with RNA-seq to profile the effects of perturbations on enhancer activity and target gene expression. We applied this approach to a library of 253,632 fragments representing 46,142 cell type–specific candidate enhancers and assessed the impact of CRISPR/Cas9-mediated deletion of six transcription factors (or TFs; ATF2, CTCF, FOXA1, LEF1, TCF7L2, and SCRT1) with diverse regulatory roles. Across knockout lines, we identified responsive enhancers that were either repressed or induced, often through motifs such as the p53 family of TFs. Enhancer–gene mapping revealed TF-specific programs, including repression of Wnt/p53 targets with ATF2 or LEF1 loss, downregulation of the FIRRE locus with CTCF loss, and compensatory upregulation of RNA polymerase II regulators following FOXA1 depletion. A deep learning model trained on enhancer sequences recapitulated core principles of enhancer grammar, including cooperative motif syntax and the influence of flanking sequence context. Applying this framework to the neurodevelopmental disorder-associated 16p12.1 deletion identified responsive enhancers linked to genes involved in axon guidance, synaptic plasticity, and translational control, providing a scalable readout of enhancer dynamics generalizable to genetic mutations.
Maitreya Das, Deepro Banerjee, Ayaan Hossain, Matthew Jensen, Saie Mogre, Jiawan Sun, Joseph Mao, Adam B. Glick, Howard M. Salis, Santhosh Girirajan. Quantifying the impact of genetic mutations on enhancer dynamics. 2025-10-21. https://doi.org/10.1101/2025.10.21.683558
A targeted library of candidate enhancer regions was assayed in HEK293T cells carrying a stable CRISPR/Cas9-mediated CTCF deletion, with three biological replicates and a matched CRISPR control (CC). The table reports fragment-level reporter activity, peak status, DESeq2 differential activity versus CC, and linked target-gene expression statistics where supplied.
The same targeted enhancer library was assayed in HEK293T cells carrying a CRISPR/Cas9-generated approximately 500-kbp 16p12.1 deletion, with three biological replicates and a matched CRISPR control (CC). The table reports fragment-level reporter activity, peak status, DESeq2 differential activity versus CC, and linked target-gene expression statistics where supplied.
A targeted library of candidate enhancer regions was assayed in HEK293T cells carrying a stable CRISPR/Cas9-mediated TCF7L2 deletion, with three biological replicates and a matched CRISPR control (CC). The table reports fragment-level reporter activity, peak status, DESeq2 differential activity versus CC, and linked target-gene expression statistics where supplied.
A targeted library of candidate enhancer regions was assayed in HEK293T cells carrying a stable CRISPR/Cas9-mediated LEF1 deletion, with three biological replicates and a matched CRISPR control (CC). The table reports fragment-level reporter activity, peak status, DESeq2 differential activity versus CC, and linked target-gene expression statistics where supplied.
A targeted library of candidate enhancer regions was assayed in HEK293T cells carrying a stable CRISPR/Cas9-mediated SCRT1 deletion, with three biological replicates and a matched CRISPR control (CC). The table reports fragment-level reporter activity, peak status, DESeq2 differential activity versus CC, and linked target-gene expression statistics where supplied.
A targeted library of candidate enhancer regions was assayed in HEK293T cells carrying a stable CRISPR/Cas9-mediated FOXA1 deletion, with three biological replicates and a matched CRISPR control (CC). The table reports fragment-level reporter activity, peak status, DESeq2 differential activity versus CC, and linked target-gene expression statistics where supplied.
A targeted library of candidate enhancer regions was assayed in HEK293T cells carrying a stable CRISPR/Cas9-mediated ATF2 deletion, with three biological replicates and a matched CRISPR control (CC). The table reports fragment-level reporter activity, peak status, DESeq2 differential activity versus CC, and linked target-gene expression statistics where supplied.
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