Biology subjects

Ornelas, I. J.

Publications and source records attributed to Ornelas, I. J..

5 recordsLinked to original sources

bioRxiv2026

Programmable CRISPRtune dissects the transcriptional repressive activity of MeCP2

The ability to control the expression of human genes is a major goal in synthetic biology, enables dissection of gene function, and can be harnessed for therapeutic applications. Advances in genome editing and transcriptional engineering often result in complete gene inactivation or full transcriptional repression. However, programmable tools to dial transcription at intermediate levels remain challenging. Here, we present CRISPRtune - a synthetic fusion of MeCP2 to catalytically dead dCas9 that tunes down transcription of endogenous genes in human cells by harnessing the mild repressor activity of MeCP2. Using pooled genome-scale CRISPR screens, we tune the expression of thousands of endogenous genes and define the targeting rules of CRISPRtune in human cells. With a platform to target MeCP2 at defined genomic sites, we show the direct epigenetic changes induced by MeCP2 at gene promoters and we identify its genetic dependency partners for productive transcriptional repression. Rett syndrome-associated mutations of MeCP2 show defects for transcriptional repression due to their failure to remodel the local epigenetic landscape of target genes. Together, we present a programmable method for transcriptional tuning in mammalian cells and offer an orthogonal platform to dissect the mechanistic function of chromatin regulators in living cells.

genomics↗

bioRxiv2026

A toolkit for programmable transcriptional engineering across eukaryotic kingdoms

Chromatin is essential for eukaryotic life. Tens of thousands of chromatin regulator (CR) proteins exist in eukaryotic genomes that are predicted to modulate chromatin states; however, their molecular functions remain largely untested experimentally. Here, we construct a library of over 300 full-length CRs from humans, plants, yeast, protozoa, and virus, each fused to DNA-binding domains, and test their direct effect on transcriptional repression and activation in plants and human cells. We discover CRs with cross-kingdom functionality when transferred across eukaryotes, including CRs that outperform existing tools for programmable transcriptional repression and activation in plants and human cells. Using pooled CRISPR screens, we demonstrate a suite of CRISPR repressors that titrate gene expression at intermediate levels. Finally, we identify RCOR1 and MTA2 as universal eukaryotic repressors that retain repressive activity in plants, yeast, and human cells. Our toolkit advances synthetic eukaryotic engineering and expands our understanding of CR functionality across eukaryotes.

synthetic biology↗

bioRxiv2024

Programmable epigenome editing by transient delivery of CRISPR epigenome editor ribonucleoproteins

Programmable epigenome editors modify gene expression in mammalian cells by altering the local chromatin environment at target loci without inducing DNA breaks. However, the large size of CRISPR-based epigenome editors poses a challenge to their broad use in biomedical research and as future therapies. Here, we present Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins (RENDER) for transiently delivering programmable epigenetic repressors (CRISPRi, DNMT3A-3L-dCas9, CRISPRoff) and activator (TET1-dCas9) as ribonucleoprotein complexes into human cells to modulate gene expression. After rational engineering, we show that RENDER induces durable epigenetic silencing of endogenous genes across various human cell types, including primary T cells. Additionally, we apply RENDER to epigenetically repress endogenous genes in human stem cell-derived neurons, including the reduction of the neurodegenerative disease associated V337M-mutated Tau protein. Together, our RENDER platform advances the delivery of CRISPR-based epigenome editors into human cells, broadening the use of epigenome editing in fundamental research and therapeutic applications.

genetics↗

bioRxiv2024

CRISPRoff epigenetic editing for programmable gene silencing in human cells without DNA breaks

The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.

synthetic biology↗

bioRxiv2022

The trans-regulatory landscape of gene networks in plants

The effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative effector domains of over 400 Arabidopsis thaliana transcription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how newly characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems.

plant biology↗