Publication Highlights

Introducing RAEFISH (Reverse-padlock Amplicon Encoding Fluorescent In Situ Hybridization), a cutting-edge technology developed by Siyuan (Steven) Wang at Yale University that transforms how we visualize and understand gene expression. Dr. Wang highlights this innovative technology that was published in Cell.

This powerful method enables whole-transcriptome imaging of over 20,000 RNA species at single-molecule resolution in cells and tissues. RAEFISH is the first method to combine high genomic coverage with high spatial resolution, revealing subcellular transcript localization, cell-type-specific patterns, and dynamic cell interactions. In addition, it also unlocks direct detection of guide RNAs for high-content CRISPR screens, paving the way for deeper insights into genetic perturbations and cellular function.

Read the full paper in Cell: https://www.cell.com/cell/fulltext/S0092-8674(25)01037-2
Data and code availability: https://campuspress.yale.edu/wanglab/RAEFISH/
Learn more about the exciting work being conducted in Dr. Siyuan (Steven) Wang’s lab: https://campuspress.yale.edu/wanglab/

Unlocking the Secrets of Pioneer Transcription Factors | Featuring Luca Mariani and Martha Bulyk
Recently published in Nature Structural & Molecular Biology, this research introduces PIONEAR-seq, a high-throughput biochemical assay that reveals how transcription factors interact with nucleosomes, the tightly packed structures of DNA and histones that regulate gene accessibility.
Transcription factors are proteins that bind to specific DNA sequences to control gene expression. Some of these factors, known as pioneers, have the unique ability to access DNA wrapped within nucleosomes, making them essential for initiating changes in cell identity and function. However, not all DNA binding sites are equally accessible, and the rules governing this accessibility have remained unclear.
Using PIONEAR-seq, the authors discovered that the broader DNA sequence context within nucleosomes plays a critical role in determining where transcription factors can bind. Surprisingly, many transcription factors show different binding behaviors on synthetic versus genomic DNA sequences, challenging long-held assumptions about pioneer factor activity.Their findings show that DNA bendability and nucleosome composition influence whether a transcription factor binds at the center or edge regions of the nucleosome. These insights challenge previous assumptions and suggest that DNA flexibility is a key regulatory feature in the genome.
Read the full paper in Nature Structural & Molecular Biology: https://www.nature.com/articles/s41594-025-01633-2
Code Availability: https://github.com/BulykLab/nucleosome_end_binding_TFs
More exciting research from the Bulyk Lab: http://the_brain.bwh.harvard.edu

Martha Bulyk, PhD and Shubham Khetan, PhD discuss their publication in Nature, titled “Multiple overlapping binding sites determine transcription factor occupancy.” This work introduces PADIT-seq, a powerful new method that maps transcription factor (TF) binding preferences across every possible 10-base-pair DNA sequence. Their findings reveal hundreds of previously undetected lower-affinity binding sites that play a crucial role in regulating gene expression. The researchers explain how traditional technologies often miss these subtle but important binding sites. They describe how overlapping DNA sequences, especially those flanking high-affinity sites, influence TF occupancy in living cells. This leads to a new model of TF binding, where occupancy is determined not by a single site but by the combined effect of multiple overlapping sites. The implications are profound, especially for understanding how noncoding genetic variants can impact gene expression and contribute to human traits and diseases.

Read the full paper in Nature: https://www.nature.com/articles/s41586-025-09472-3
Code and processed data for generating the figures are available at https://github.com/BulykLab/PADIT-seq
Discover more from the Bulyk Lab: http://thebrain.bwh.harvard.edu/

What if DNA didn’t use A, C, G, or T? Andrew Laszlo and Christopher Thomas describe how they were able to successfully sequence a synthetic DNA molecule made from four artificial bases (P, Z, S, and B) using a custom nanopore setup. This “ALIEN DNA”, , designed by FfAME – Foundation for Applied Molecular Evolution, mimics the structure of natural DNA but is completely orthogonal to biological systems, making it ideal for applications in synthetic biology, molecular diagnostics, and even astrobiology. Using variable-voltage nanopore sequencing, the team achieved single-molecule, label-free reads of these artificial strands with impressive accuracy. This breakthrough shows that nanopore technology can be adapted to decode entirely new genetic alphabets, opening the door to designing and reading custom genetic systems.

Read the full paper in Nature Communications: https://doi.org/10.1038/s41467-025-61991-9
The analysis code are available: https://doi.org/10.6084/m9.figshare.28199681.v2
Learn more about the NHGRI Genome Technology Opportunity Fund: https://genometdcc.org/opportunity-fund-program/
UW Nanopore Biophysics Lab: https://depts.washington.edu/nanopore/index.html
Foundation for Applied Molecular Evolution: https://www.ffame.org/home.php

The 3D organization of our genome plays a crucial role in development, aging, and disease—but what controls this complex spatial folding of DNA remains largely mysterious. In this video, Dr. Steven Wang and Yubao Cheng of Yale University unveil a groundbreaking platform called Perturb-tracing that enables large-scale discovery of chromatin topology regulators at multiple length scales. Published in Nature Methods. This new image-based screening method combines:

🔹 Pooled CRISPR-based gene perturbations
🔹 A powerful barcode readout system (BARC-FISH)
🔹 High-resolution chromatin tracing within single cells

Using this platform, they performed loss-of-function screens in human cells to visualize how 3D chromatin structures—from local domains to entire chromosome territories—change with specific gene knockouts. The result? They discovered dozens of new regulators involved in chromatin folding, some tied to known mechanisms like loop extrusion and A/B compartmentalization, while others revealed unexpected pathways and nuclear architecture influences.

Key Highlights:
🔹 First scalable method to screen 3D genome organization across multiple scales
🔹 Identification of novel genes affecting chromatin compaction and nuclear shape
🔹 Insights into regulatory mechanisms beyond classical chromatin models

Whether you’re into genomics, cellular architecture, or high-content screening technologies, this research opens up powerful new avenues for understanding how the genome’s 3D shape affects its function.
Learn more about the work from Dr. Wang’s lab: https://campuspress.yale.edu/wanglab/
Read the paper in Nature Methods: https://www.nature.com/articles/s41592-025-02652-z
Data Collection: Open-source codes for imaging data collection are available at https://github.com/ZhuangLab/storm-control/
Data Analysis: All orginal codes generated for this study are available at https://campuspress.yale.edu/wanglab/BARCFISH/ and https://doi.org/10.5281/zenodo.14227128/

Revolutionizing Spatial Transcriptomics: New Imaging-Free Method!

Spatial transcriptomics maps gene expression data to specific locations in a tissue section, revealing cellular heterogeneity, spatial organization, and functional interactions. Traditional methods require specialized imaging to track RNA molecules within tissues or indexed capture arrays. In this publication highlight, Fei Chen, Ph.D., and Chenlei Hu of Broad Institute of Harvard and MIT, discuss a new, imaging-free spatial transcriptomics approach that opens the door for high-throughput, cost-effective, and large-scale studies without the need for expensive setups. This new method uses dimensionality reduction to computationally reconstruct array barcode locations used in spatial transcriptomics measurements with high resolution and fidelity. Published in Nature Biotechnology.
Read the paper in Nature Biotechnology: https://www.nature.com/articles/s41587-025-02612-0
Code available: http://github.com/Chenlei-Hu/Slide_recon.git
Fei Chen Lab: https://www.insitubiology.org/

🔬 Key Topics:

• Structural variants (SVs) in mammalian genomes
• Genome-Shuffle-seq: A new method for mapping and characterizing SVs
• Functional consequences of SVs in mouse and human cells
• Impacts of SVs on gene expression and cellular fitness

Sudarshan Pinglay, PhD, introduces Genome-Shuffle-seq, a method developed to revolutionize the study of structural variants (SVs) in mammalian genomes (read the article “Multiplex generation and single-cell analysis of structural variants in mammalian genomes” in Science https://doi.org/10.1126/science.ado5978). SVs—such as deletions, inversions, duplications, translocations, and extrachromosomal DNA circles—are known to play a crucial role in both rare and common diseases, as well as in normal genetic variation. However, studying these complex variations at scale has been a significant challenge—until now.

Learn about how Genome-Shuffle-seq enables the multiplex generation, mapping, and characterization of several major SV classes using an innovative approach. This method leverages barcoded “shuffle cassettes” for genome-wide mapping, site-specific recombination, and novel barcode pairings that reveal the breakpoints and class of each SV.

Genome-Shuffle-seq not only provides a high-resolution, cost-effective approach for SV mapping across the entire human genome but also enables large-scale, cellular screens to quantify SV impacts on fitness, gene expression, chromatin state, and 3D nuclear architecture. These data are crucial for understanding the functional architecture of mammalian genomes and could ultimately advance our knowledge of SVs associated with human phenotypes.

Learn more about the Pinglay lab: https://www.pinglay-lab.com/

📚 Related research article mentioned in the video:

Randomizing the human genome by engineering recombination between repeat elements Genome mapping techniques. J. Koeppel et al., Science 387, eado3979(2025). https://doi.org/10.1126/science.ado3979

Explore groundbreaking advancements in protein sequencing with this video on multi-pass, single-molecule nanopore technology! Jeff Nivala, Ph.D., Keisuke Motone, Ph.D., and Daphne Kontogiorgos-Heintz discuss the development of a method to read intact, long protein strands using nanopore sensors, overcoming limitations of current technologies like Edman degradation and mass spectrometry. By leveraging ClpX unfoldase to pull proteins through a CsgG nanopore on the Oxford Nanopore Technologies MinION R9.4 flow cell, Nivala’s group achieved single-molecule sensitivity to amino acids, allowing for the detection of post-translational modifications such as phosphorylation. This innovative approach paves the way for accurate protein barcoding, identification of proteoforms, and deeper insights into biological processes and disease mechanisms. Watch now to learn how nanopore technology is revolutionizing proteomics!
Key Highlights:
• Full-length protein sequencing of designed proteins using nanopores.
• Multi-pass rereading of protein molecules for increased accuracy.
• Detection of single amino acid substitutions and post-translational modifications.
• Potential applications in proteoform identification and therapeutic development.
For more details, read the full article on Nature: https://doi.org/10.1038/s41586-024-07935-7
Code for analyses is available on Github: https://github.com/uwmisl/PASTOR-sequencing
Custom MinION MinKNOW run scripts can be obtained from Oxford Nanopore Technologies on request.
Jeff Nivala website: https://www.jeffnivala.com/

Nicholas Drachman and Derek Stein, Ph.D. of Brown University, discuss their innovative development in mass spectrometry-based proteomics, published in Nature Communications. They developed custom mass spectrometry nanopore ion sources to deliver single ions of individual amino acids and peptides directly into high vacuum from biologically relevant aqueous solutions. This work makes significant improvements over conventional electrospray ionization methods. This is a huge step forward for mass spectrometry—a technique used to measure the mass of molecules—and its significance in proteomics, where understanding protein structure and function is critical for advancing biology and medicine.
Read the full paper in Nature Communications: https://doi.org/10.1038/s41467-024-51455-x

Alejandro Chavez, MD, PhD, and Joonwon Kim, PhD of UCSD discuss their recent Science Advances publication on high-throughput insertion of tags across the genome (HITAG). This cutting-edge technique enables rapid and efficient tagging of endogenous proteins, providing an invaluable tool for the large-scale interrogation of protein function.

What is HITAG? HITAG (High-Throughput Insertion of Tags Across the Genome) is a novel method designed to facilitate the study of protein function on a large scale. By leveraging a modified Cas9-based targeted insertion strategy that relies on nonhomologous end joining (NHEJ), HITAG allows for the rapid creation of libraries of cells, each containing a different protein of interest tagged at the C-terminus.

Applications: Using HITAG, the researchers fused mCherry to a set of 167 stress granule-associated proteins. This enabled them to elucidate the features driving a subset of proteins to accumulate strongly within these transient RNA-protein granules.

Why is HITAG Important? Understanding the dynamic behavior and interaction partners of proteins is crucial for building an accurate working model of the cell. Protein tags, such as those created by HITAG, facilitate numerous studies, including in vivo protein localization, affinity purification, and rapid protein degradation.

Read the full article in Science Advances: https://www.science.org/doi/10.1126/sciadv.adg8771
Plasmid constructs available at the nonprofit repository, Addgene:  https://www.addgene.org/browse/article/28238585/
Learn more about the Chavez Lab and research interests: https://chavezlab.com/

Pre-mRNA splicing is an important step in gene expression. Antisense oligonucleotides (ASOs) can therapeutically modulate splicing by competing against endogenous splicing factors. The ASO therapeutic Spinraza/nusinersen, is the first efficacious treatment for spinal muscular atrophy (SMA), targeting an intronic splicing silencer downstream of Survival Motor Neuron 2 (SMN2) exon 7, rescuing SMN protein production in SMA patients. The process for discovering ASO targets is time intensive and expensive. In this publication highlight, Chaolin Zhang, Ph.D., and Yow-Tyng Yeh, Ph.D., of Columbia University discuss SpliceRUSH, a high-throughput screening method that uses a CRISPR-dCas13d RNA-target system that exploits the similar mode of action of ASOs to uncover proximal and distal splicing-regulatory elements (SREs). When applied to SMN2, SpliceRUSH identifies not only the known therapeutic targets, but also uncovers a previously unknown distal intronic SRE. *Read the full paper here* https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11076525/ *Visit the Chaolin Zhang Lab* https://zhanglab.c2b2.columbia.edu/index.php/ *Code Availability* The scripts used for this study are available at https://github.com/chaolinzhanglab/rush

Hugo Medina-Muñoz, PhD and Gene Yeo, PhD, MBA (University of California, San Diego) highlight the work published in Nature Communications describing an experimental and computational framework called PRINTER (protein-RNA-interaction-based triaging of enzymes that edit RNA). RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Several methods have been established to identify RNA targets of RBPs including antibody-free methods that use RNA base-editors (rBEs) fused to RBPs. The PRINTER framework expands the repertoire of available base editors and provides a rapid means to characterize these enzymes before their application in RBP-mediated editing. It also enables the identification of DNA editors, addressing a critical aspect of the field’s current limitations. The study emphasizes the importance of acknowledging and addressing enzyme bias when designing experiments and interpreting their outcomes. Relying solely on one enzyme can lead to false negatives and an incomplete understanding of protein-RNA interactions.
Access the full, free text of the Nature Communications publication https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10825223/

Code Availability
The software utilized and code generated for the analyses in this study can be obtained by accessing the following repositories:
snakemake
GitHub: PRINTER / FLARE

Visit Gene Yeo Lab for more details on this and other projects.
This work was partially supported by an NHGRI Opportunity Fund to Gene W. Yeo and Rahul M. Kohli.
Opportunity Funds are administered by the TDCC and provided as a subcontract from NIH grant U24HG011735.

Combining the high-quality and high-throughput quantification of single-nucleus genomics measurements with the spatial context of tissue measurements comes Slide-tags, an innovative method in which single nuclei in intact tissue sections are tagged with spatial barcode oligonucleotides derived from DNA-barcode beads of known position. Hear from Andrew Russell, Jackson Weir, Naeem Nadaf, and Fei Chen as they describe the development and application of this impressive technology that only adds 10 minutes to existing single-nucleus RNA sequencing workflows.

Read the article published in Nature: https://www.nature.com/articles/s41586-023-06837-4

Code availability:
Code for processing spatial sequencing libraries is available at GitHub https://github.com/broadchenf/Slide-tags (https://doi.org/10.5281/zenodo.2571615)

Fei Chen Lab: https://www.insitubiology.org/
Evan Macosko Lab: https://macoskolab.com/

RNA-binding proteins (RBPs) mediate various cellular processes and play a major role in post-translational gene regulation of RNAs, including splicing. There are over 2000 predicted RBPs, many without an assigned RNA-binding function. Gene Yeo, PhD, MBA (University of California, San Diego) and Jonathan Schmok, PhD (The Arc Institute) discuss development of tethered function splicing reporter assays to evaluate over 700 RBPs in their latest paper published in Nature Biotechnology (https://doi.org/10.1038/s41467-024-45009-4). Hear how they performed enhanced cross-linking immoprecipitation (eCLIP), knockdown RNA sequencing, and affinity purification mass spectrometry to investigate candidates with no prior association with RNA splicing. Visit The Yeo Lab to learn more!
RNA-Binding Protein Analysis & Repository Tool Kit
Skipper – Computational analysis of eCLIP data on GitHub

Small nucleolar (sno)RNAs are a large family of noncoding RNAs that often use antisense guide sequences to recognize target RNAs. The vast majority of targets for the ~2000 snoRNAs in humans remains unknown. Zhipeng Lu, Ph.D., and co-lead authors Minjie Zhang, Ph.D., and Kongpan Li, Ph.D., discuss improvements to genomic technologies that led to the identification and verification of a large network of snoRNA-tRNA interactions. These findings reveal a supply-and-demand economy where snoRNAs regulate tRNA usage to influence cell proliferation and development. Application of the improved PARIS2 and denatured RiboMeth-seq methods may allow dissection of other noncoding RNAs’ function in the human genome. Visit Zhipeng Lu’s Lab to learn more about work being conducted.

Read the full paper published in Proc Natl Acad Sci USA. 2023; 120 (41):e231216120..

COVID-19 infections caused by SARS-CoV-2, a coronavirus, are still prevalent worldwide. Having multiple targets available to design antivirals, especially to highly conserved genes across coronaviruses, is ideal. The SARS-CoV-2 helicase, nsp13, is essential for viral replication and is highly conserved. Understanding the mechanics of nsp13 action is difficult due to the speed and small step size of the motor enzyme. Dr. Andrew Laszlo (University of Washington) and Dr. Sinduja Marx (Seattle Children’s Research Institute) discuss the use of Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) to reveal the detailed mechanism of nsp13 motion on DNA and demonstrate how SPRNT can be used to determine mechanism of action of a helicase inhibitor.

Read more about this published work in Nucleic Acids Research. 2023; 51(17):9266-9278.

Nanopore technology has transformed how the world sequences DNA and RNA enabling a deeper understanding of genomes. The next frontier for nanopores will be protein sequencing. Giovanni Maglia, Ph.D. and Adina Sauciuc, MSc. (University of Groningen) discuss how they engineered a nanopore to translocate untagged, linearized, full-length proteins under an opposing electroosmotic force and achieve unique protein signatures. Visit Giovanni Maglia’s lab to learn more about single-molecule biophysics.

Read the full paper published in Nat Biotechnol (2023) https://doi.org/10.1038/s41587-023-01954-x

Drs. Rahul M. Kohli and Tong Wang discuss a new non-destructive enzymatic sequencing method for detecting DNA methylation that they developed called Direct Methylation Sequencing (DM-Seq). Learn about the technique that allows scientists to profile very small quantities of DNA to clearly identify 5-methylcytosine at base-resolution. Visit Rahul Kohli’s lab to learn more about exciting innovations and current projects.

Read the full paper published in Nat Chem Biol 2023; 19(8):1004-1012.