Biology subjects

Pattali, R. K.

Publications and source records attributed to Pattali, R. K..

4 recordsLinked to original sources

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↗

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↗