Biology subjects

Lubin, J. P.

Publications and source records attributed to Lubin, J. P..

3 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

LEMONmethyl-seq: Targeted long-read DNA methylation profiling reveals dynamics of CRISPR epigenome editing and endogenous DNA methylation patterns

BACKGROUNDDNA methylation is the most prevalent epigenetic modification in human cells and undergoes dynamic changes during cell differentiation, disease progression, and aging. Here, we introduce Locus-Enriched Mapping Of Nucleotide methylation (LEMONmethyl-seq): an optimized, cost-effective pipeline for single-nucleotide detection of DNA methylation using locus-specific amplification and long-read DNA sequencing. RESULTSWe apply LEMONmethyl-seq to profile DNA methylation of endogenous gene promoters across different cell types along with DNA methylation establishment and long-range propagation induced by CRISPR epigenome editing technologies. We profile dynamic changes in DNA methylation patterns on transposable element genomic loci during global epigenetic resetting in stem cells, and we identify site-specific enrichment of non-canonical CpH methylation on genomic sites in stem cells and cultured neurons. Lastly, we apply LEMONmethyl-seq to profile DNA methylation across the MGMT promoter, a clinical biomarker for glioblastoma. We identify additional differentially methylated sites correlated with chemotherapeutic sensitivity, which may be clinically relevant. CONCLUSIONSTogether, LEMONmethyl-seq serves as a cost-effective, long-read DNA methylation sequencing pipeline that advances methods for detecting DNA methylation patterns and dynamics in mammalian cells. We envision its broad use for studying chromatin pathways, diagnostics, and therapeutic applications.

molecular 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↗