Recent Publications

Publications associated with the Genome Technologies program.

Recent Publications2023-09-07T09:20:11-04:00

Click here to see Grantee’s published work – available via PubMed.

Modular pooled discovery of synthetic knockin sequences to program durable cell therapies
Publication: Cell
Date: Fri, 15 Sep 2023
Authors: Franziska Blaeschke, Yan Yi Chen, Ryan Apathy, Bence Daniel, Andy Y Chen, Peixin Amy Chen, Katalin Sandor, Wenxi Zhang, Zhongmei Li, Cody T Mowery, Tori N Yamamoto, William A Nyberg, Angela To, Ruby Yu, Raymund Bueno, Min Cheol Kim, Ralf Schmidt, Daniel B Goodman, Tobias Feuchtinger, Justin Eyquem, Chun Jimmie Ye, Julia Carnevale, Ansuman T Satpathy, Eric Shifrut, Theodore L Roth, Alexander Marson,
Identifier: pmid:37714135
Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors......Read more
Transcriptomic profiling of tissue environments critical for post-embryonic patterning and morphogenesis of zebrafish skin
Publication: eLife
Date: Mon, 11 Sep 2023
Authors: Andrew J Aman, Lauren M Saunders, August A Carr, Sanjay Srivatasan, Colten Eberhard, Blake Carrington, Dawn Watkins-Chow, William J Pavan, Cole Trapnell, David M Parichy,
Identifier: pmid:37695017
Pigment patterns and skin appendages are prominent features of vertebrate skin. In zebrafish, regularly patterned pigment stripes and an array of calcified scales form simultaneously in the skin during post-embryonic development. Understanding the mechanisms that regulate stripe patterning and scale morphogenesis may lead to the discovery of fundamental mechanisms that govern the development of animal form. To learn about cell types and signaling interactions that govern skin patterning and......Read more
Single-cell sequencing of individual retinal organoids reveals determinants of cell-fate heterogeneity
Publication: Cell reports methods
Date: Wed, 06 Sep 2023
Authors: Amy Tresenrider, Akshayalakshmi Sridhar, Kiara C Eldred, Sophia Cuschieri, Dawn Hoffer, Cole Trapnell, Thomas A Reh,
Identifier: pmid:37671011
With a critical need for more complete in vitro models of human development and disease, organoids hold immense potential. Their complex cellular composition makes single-cell sequencing of great utility; however, the limitation of current technologies to a handful of treatment conditions restricts their use in screens or studies of organoid heterogeneity. Here, we apply sci-Plex, a single-cell combinatorial indexing (sci)-based RNA sequencing (RNA-seq) multiplexing method to retinal organoids.......Read more
Deep screening of proximal and distal splicing-regulatory elements in a native sequence context
Publication: bioRxiv : the preprint server for biology
Date: Mon, 04 Sep 2023
Authors: Yocelyn Recinos, Dmytro Ustianenko, Yow-Tyng Yeh, Xiaojian Wang, Martin Jacko, Lekha V Yesantharao, Qiyang Wu, Chaolin Zhang,
Identifier: pmid:37662340
Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is impeded by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically dead CRISPR- Rfx Cas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against......Read more
A molecular proximity sensor based on an engineered, dual-component guide RNA
Publication: bioRxiv : the preprint server for biology
Date: Wed, 30 Aug 2023
Authors: Junhong Choi, Wei Chen, Hanna Liao, Xiaoyi Li, Jay Shendure,
Identifier: pmid:37645782
One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events......Read more
Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects
Publication: Nature biotechnology
Date: Mon, 28 Aug 2023
Authors: Emily B Fabyanic, Peng Hu, Qi Qiu, Kiara N Berríos, Daniel R Connolly, Tong Wang, Jennifer Flournoy, Zhaolan Zhou, Rahul M Kohli, Hao Wu,
Identifier: pmid:37640946
Oxidative modification of 5-methylcytosine (5mC) by ten-eleven translocation (TET) DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here, we present joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously......Read more
The complete sequence of a human Y chromosome
Publication: Nature
Date: Wed, 23 Aug 2023
Authors: Arang Rhie, Sergey Nurk, Monika Cechova, Savannah J Hoyt, Dylan J Taylor, Nicolas Altemose, Paul W Hook, Sergey Koren, Mikko Rautiainen, Ivan A Alexandrov, Jamie Allen, Mobin Asri, Andrey V Bzikadze, Nae-Chyun Chen, Chen-Shan Chin, Mark Diekhans, Paul Flicek, Giulio Formenti, Arkarachai Fungtammasan, Carlos Garcia Giron, Erik Garrison, Ariel Gershman, Jennifer L Gerton, Patrick G S Grady, Andrea Guarracino, Leanne Haggerty, Reza Halabian, Nancy F Hansen, Robert Harris, Gabrielle A Hartley, William T Harvey, Marina Haukness, Jakob Heinz, Thibaut Hourlier, Robert M Hubley, Sarah E Hunt, Stephen Hwang, Miten Jain, Rupesh K Kesharwani, Alexandra P Lewis, Heng Li, Glennis A Logsdon, Julian K Lucas, Wojciech Makalowski, Christopher Markovic, Fergal J Martin, Ann M Mc Cartney, Rajiv C McCoy, Jennifer McDaniel, Brandy M McNulty, Paul Medvedev, Alla Mikheenko, Katherine M Munson, Terence D Murphy, Hugh E Olsen, Nathan D Olson, Luis F Paulin, David Porubsky, Tamara Potapova, Fedor Ryabov, Steven L Salzberg, Michael E G Sauria, Fritz J Sedlazeck, Kishwar Shafin, Valery A Shepelev, Alaina Shumate, Jessica M Storer, Likhitha Surapaneni, Angela M Taravella Oill, Françoise Thibaud-Nissen, Winston Timp, Marta Tomaszkiewicz, Mitchell R Vollger, Brian P Walenz, Allison C Watwood, Matthias H Weissensteiner, Aaron M Wenger, Melissa A Wilson, Samantha Zarate, Yiming Zhu, Justin M Zook, Evan E Eichler, Rachel J O'Neill, Michael C Schatz, Karen H Miga, Kateryna D Makova, Adam M Phillippy,
Identifier: pmid:37612512
The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure that includes long palindromes, tandem repeats and segmental duplications^(1-3). As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished^(4,5). Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029-base-pair sequence of a human Y chromosome from the HG002......Read more
C-Terminal Arginine-Selective Cleavage of Peptides as a Method for Mimicking Carboxypeptidase B
Publication: Organic letters
Date: Wed, 16 Aug 2023
Authors: Lyndsey C Prosser, John M Talbott, Rose P Garrity, Monika Raj,
Identifier: pmid:37585337
C-Terminal residues play a pivotal role in dictating the structure and functions of proteins. Herein, we report a mild, efficient, chemoselective, and site-selective chemical method that allows for precise chemical proteolysis at C-terminal arginine dictated by 9,10-phenanthrenequinone independent of the remaining sequence. This biomimetic approach also exhibits the potential to synthesize C-terminal methyl ester (-CO(2)Me) peptides....Read more
Genome-wide prediction of disease variant effects with a deep protein language model
Publication: Nature genetics
Date: Thu, 10 Aug 2023
Authors: Nadav Brandes, Grant Goldman, Charlotte H Wang, Chun Jimmie Ye, Vasilis Ntranos,
Identifier: pmid:37563329
Predicting the effects of coding variants is a major challenge. While recent deep-learning models have improved variant effect prediction accuracy, they cannot analyze all coding variants due to dependency on close homologs or software limitations. Here we developed a workflow using ESM1b, a 650-million-parameter protein language model, to predict all ~450 million possible missense variant effects in the human genome, and made all predictions available on a web portal. ESM1b outperformed......Read more
Observing inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution
Publication: Nucleic acids research
Date: Thu, 10 Aug 2023
Authors: Sinduja K Marx, Keith J Mickolajczyk, Jonathan M Craig, Christopher A Thomas, Akira M Pfeffer, Sarah J Abell, Jessica D Carrasco, Michaela C Franzi, Jesse R Huang, Hwanhee C Kim, Henry Brinkerhoff, Tarun M Kapoor, Jens H Gundlach, Andrew H Laszlo,
Identifier: pmid:37560916
The genome of SARS-CoV-2 encodes for a helicase (nsp13) that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds double-stranded DNA. Our data reveal nsp13's......Read more
A genome-wide single-cell 3D genome atlas of lung cancer progression
Publication: bioRxiv : the preprint server for biology
Date: Mon, 07 Aug 2023
Authors: Miao Liu, Shengyan Jin, Sherry S Agabiti, Tyler B Jensen, Tianqi Yang, Jonathan S D Radda, Christian F Ruiz, Gabriel Baldissera, Mandar Deepak Muzumdar, Siyuan Wang,
Identifier: pmid:37546882
Alterations in three-dimensional (3D) genome structures are associated with cancer ^(1-5) . However, how genome folding evolves and diversifies during subclonal cancer progression in the native tissue environment remains unknown. Here, we leveraged a genome-wide chromatin tracing technology to directly visualize 3D genome folding in situ in a faithful Kras -driven mouse model of lung adenocarcinoma (LUAD) ⁶ , generating the first single-cell 3D genome atlas of any cancer. We discovered......Read more
Multivalent binding of the tardigrade Dsup protein to chromatin promotes yeast survival and longevity upon exposure to oxidative damage
Publication: Research square
Date: Mon, 07 Aug 2023
Authors: Jessica Tyler, Rhiannon Aguilar, Nina Arslanovic, Kaylah Birmingham, Kritika Kaliwal, Ujani Chakraborty, Spike Postnikoff, Allison Hickman, Laiba Kahn, Rachel Watson, Ryan Ezell, Hannah Willis, Martis Cowles, Matthew Marunde, Michael-C Keogh, Ignacio Gutierrez, Abraham Shim, Richard Garner,
Identifier: pmid:37546815
Tardigrades are remarkable in their ability to survive extreme environments. The damage suppressor (Dsup) protein is thought responsible for their extreme resistance to reactive oxygen species (ROS) generated by irradiation. Here we show that expression of Ramazzottius varieornatus Dsup in Saccharomyces cerevisiae reduces oxidative DNA damage and extends the lifespan of budding yeast exposed to chronic oxidative genotoxicity. This protection from ROS requires either the Dsup HMGN-like domain or......Read more
High-throughput functional characterization of combinations of transcriptional activators and repressors
Publication: Cell systems
Date: Sat, 05 Aug 2023
Authors: Adi X Mukund, Josh Tycko, Sage J Allen, Stephanie A Robinson, Cecelia Andrews, Joydeb Sinha, Connor H Ludwig, Kaitlyn Spees, Michael C Bassik, Lacramioara Bintu,
Identifier: pmid:37543039
Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, whereas combining strong activators often results in......Read more
Female naïve human pluripotent stem cells carry X chromosomes with Xa-like and Xi-like folding conformations
Publication: Science advances
Date: Fri, 04 Aug 2023
Authors: Benjamin Patterson, Bing Yang, Yoshiaki Tanaka, Kun-Yong Kim, Bilal Cakir, Yangfei Xiang, Jonghun Kim, Siyuan Wang, In-Hyun Park,
Identifier: pmid:37540754
Three-dimensional (3D) genomics shows immense promise for studying X chromosome inactivation (XCI) by interrogating changes to the X chromosomes' 3D states. Here, we sought to characterize the 3D state of the X chromosome in naïve and primed human pluripotent stem cells (hPSCs). Using chromatin tracing, we analyzed X chromosome folding conformations in these cells with megabase genomic resolution. X chromosomes in female naïve hPSCs exhibit folding conformations similar to the active X......Read more
Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine
Publication: Chembiochem : a European journal of chemical biology
Date: Sun, 30 Jul 2023
Authors: Sigal Avraham, Leonie Schütz, Larissa Käver, Andreas Dankers, Sapir Margalit, Yael Michaeli, Shahar Zirkin, Dmitry Torchinsky, Noa Gilat, Omer Bahr, Gil Nifker, Maya Koren-Michowitz, Elmar Weinhold, Yuval Ebenstein,
Identifier: pmid:37518671
5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established......Read more
CRISPR/Cas9-Based Screening of FDA-Approved Drugs for NRF2 Activation: A Novel Approach to Discover Therapeutics for Non-Alcoholic Fatty Liver Disease
Publication: Antioxidants (Basel, Switzerland)
Date: Sat, 29 Jul 2023
Authors: James Li, Sandra Arest, Bartlomiej Olszowy, John Gordon, Carlos A Barrero, Oscar Perez-Leal,
Identifier: pmid:37507903
With the rising prevalence of obesity, non-alcoholic fatty liver disease (NAFLD) now affects 20-25% of the global population. NAFLD, a progressive condition associated with oxidative stress, can result in cirrhosis and liver cancer in 10% and 3% of patients suffering NAFLD, respectively. Therapeutic options are currently limited, emphasizing the need for novel treatments. In this study, we examined the potential of activating the transcription factor NRF2, a crucial player in combating oxidative......Read more
Engineering Biological Nanopore Approaches toward Protein Sequencing
Publication: ACS nano
Date: Tue, 25 Jul 2023
Authors: Xiaojun Wei, Tadas Penkauskas, Joseph E Reiner, Celeste Kennard, Mark J Uline, Qian Wang, Sheng Li, Aleksei Aksimentiev, Joseph W F Robertson, Chang Liu,
Identifier: pmid:37490313
Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies, while the methods we have for identifying and quantifying individual proteins are still inadequate to perform protein sequencing at the single-molecule level. Nanopore-inspired systems devoted to understanding how single molecules behave have been extensively developed for applications in genome sequencing. These nanopore systems are emerging as prominent tools for protein identification,......Read more
Single-molecule approaches for DNA damage detection and repair: A focus on Repair Assisted Damage Detection (RADD)
Publication: DNA repair
Date: Wed, 19 Jul 2023
Authors: Tahir Detinis Zur, Jasline Deek, Yuval Ebenstein,
Identifier: pmid:37467630
The human genome is continually exposed to various stressors, which can result in DNA damage, mutations, and diseases. Among the different types of DNA damage, single-strand lesions are commonly induced by external stressors and metabolic processes. Accurate detection and quantification of DNA damage are crucial for understanding repair mechanisms, assessing environmental impacts, and evaluating response to therapy. However, traditional techniques have limitations in sensitivity and the ability......Read more
Pre-mRNA splicing order is predetermined and maintains splicing fidelity across multi-intronic transcripts
Publication: Nature structural & molecular biology
Date: Thu, 13 Jul 2023
Authors: Karine Choquet, Autum R Baxter-Koenigs, Sarah-Luisa Dülk, Brendan M Smalec, Silvi Rouskin, L Stirling Churchman,
Identifier: pmid:37443198
Combinatorially, intron excision within a given nascent transcript could proceed down any of thousands of paths, each of which would expose different dynamic landscapes of cis-elements and contribute to alternative splicing. In this study, we found that post-transcriptional multi-intron splicing order in human cells is largely predetermined, with most genes spliced in one or a few predominant orders. Strikingly, these orders were conserved across cell types and stages of motor neuron......Read more
Multiplex enzymatic synthesis of DNA with single-base resolution
Publication: Science advances
Date: Fri, 07 Jul 2023
Authors: Damiano Verardo, Beatrice Adelizzi, Daniel A Rodriguez-Pinzon, Nicolas Moghaddam, Emma Thomée, Tessa Loman, Xavier Godron, Adrian Horgan,
Identifier: pmid:37418522
Enzymatic DNA synthesis (EDS) is a promising benchtop and user-friendly method of nucleic acid synthesis that, instead of solvents and phosphoramidites, uses mild aqueous conditions and enzymes. For applications such as protein engineering and spatial transcriptomics that require either oligo pools or arrays with high sequence diversity, the EDS method needs to be adapted and certain steps in the synthesis process spatially decoupled. Here, we have used a synthesis cycle comprising a first step......Read more
Spatiotemporal transcriptomic maps of whole mouse embryos at the onset of organogenesis
Publication: Nature genetics
Date: Thu, 06 Jul 2023
Authors: Abhishek Sampath Kumar, Luyi Tian, Adriano Bolondi, Amèlia Aragonés Hernández, Robert Stickels, Helene Kretzmer, Evan Murray, Lars Wittler, Maria Walther, Gabriel Barakat, Leah Haut, Yechiel Elkabetz, Evan Z Macosko, Léo Guignard, Fei Chen, Alexander Meissner,
Identifier: pmid:37414952
Spatiotemporal orchestration of gene expression is required for proper embryonic development. The use of single-cell technologies has begun to provide improved resolution of early regulatory dynamics, including detailed molecular definitions of most cell states during mouse embryogenesis. Here we used Slide-seq to build spatial transcriptomic maps of complete embryonic day (E) 8.5 and E9.0, and partial E9.5 embryos. To support their utility, we developed sc3D, a tool for reconstructing and......Read more
Single-cell sequencing of individual retinal organoids reveals determinants of cell fate heterogeneity
Publication: bioRxiv : the preprint server for biology
Date: Mon, 03 Jul 2023
Authors: Amy Tresenrider, Akshayalakshmi Sridhar, Kiara C Eldred, Sophia Cuschieri, Dawn Hoffer, Cole Trapnell, Thomas A Reh,
Identifier: pmid:37398481
With a critical need for more complete in vitro models of human development and disease, organoids hold immense potential. Their complex cellular composition makes single-cell sequencing of great utility; however, the limitation of current technologies to a handful of treatment conditions restricts their use in screens or studies of organoid heterogeneity. Here, we apply sci-Plex, a single-cell combinatorial indexing (sci)-based RNA-seq multiplexing method to retinal organoids. We demonstrate......Read more
Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses
Publication: bioRxiv : the preprint server for biology
Date: Mon, 03 Jul 2023
Authors: Kaiyi Jiang, Justin Lim, Samantha Sgrizzi, Michael Trinh, Alisan Kayabolen, Natalya Yutin, Eugene V Koonin, Omar O Abudayyeh, Jonathan S Gootenberg,
Identifier: pmid:37398409
TnpB proteins are RNA-guided nucleases that are broadly associated with IS200/605 family transposons in prokaryotes. TnpB homologs, named Fanzors, have been detected in genomes of some eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. We searched genomes of diverse eukaryotes and their viruses for TnpB homologs and identified numerous putative RNA-guided nucleases that are often associated with various transposases, suggesting they are encoded in mobile......Read more
Zfp697 is an RNA-binding protein that regulates skeletal muscle inflammation and regeneration
Publication: bioRxiv : the preprint server for biology
Date: Mon, 03 Jul 2023
Authors: Jorge C Correia, Paulo R Jannig, Maya L Gosztyla, Igor Cervenka, Serge Ducommun, Stine M Præstholm, Kyle Dumont, Zhengye Liu, Qishan Liang, Daniel Edsgärd, Olof Emanuelsson, Paul Gregorevic, Håkan Westerblad, Tomas Venckunas, Marius Brazaitis, Sigitas Kamandulis, Johanna T Lanner, Gene W Yeo, Jorge L Ruas,
Identifier: pmid:37398033
Muscular atrophy is a mortality risk factor that happens with disuse, chronic disease, and aging. Recovery from atrophy requires changes in several cell types including muscle fibers, and satellite and immune cells. Here we show that Zfp697/ZNF697 is a damage-induced regulator of muscle regeneration, during which its expression is transiently elevated. Conversely, sustained Zfp697 expression in mouse muscle leads to a gene expression signature of chemokine secretion, immune cell recruitment, and......Read more
Skipper analysis of eCLIP datasets enables sensitive detection of constrained translation factor binding sites
Publication: Cell genomics
Date: Fri, 30 Jun 2023
Authors: Evan A Boyle, Hsuan-Lin Her, Jasmine R Mueller, Jack T Naritomi, Grady G Nguyen, Gene W Yeo,
Identifier: pmid:37388912
Technology for crosslinking and immunoprecipitation (CLIP) followed by sequencing (CLIP-seq) has identified the transcriptomic targets of hundreds of RNA-binding proteins in cells. To increase the power of existing and future CLIP-seq datasets, we introduce Skipper, an end-to-end workflow that converts unprocessed reads into annotated binding sites using an improved statistical framework. Compared with existing methods, Skipper on average calls 210%-320% more transcriptomic binding sites and......Read more
Detection of phosphorylation post-translational modifications along single peptides with nanopores
Publication: Nature biotechnology
Date: Thu, 29 Jun 2023
Authors: Ian C Nova, Justas Ritmejeris, Henry Brinkerhoff, Theo J R Koenig, Jens H Gundlach, Cees Dekker,
Identifier: pmid:37386295
Current methods to detect post-translational modifications of proteins, such as phosphate groups, cannot measure single molecules or differentiate between closely spaced phosphorylation sites. We detect post-translational modifications at the single-molecule level on immunopeptide sequences with cancer-associated phosphate variants by controllably drawing the peptide through the sensing region of a nanopore. We discriminate peptide sequences with one or two closely spaced phosphates with 95%......Read more
High-throughput discovery and characterization of viral transcriptional effectors in human cells
Publication: Cell systems
Date: Thu, 22 Jun 2023
Authors: Connor H Ludwig, Abby R Thurm, David W Morgens, Kevin J Yang, Josh Tycko, Michael C Bassik, Britt A Glaunsinger, Lacramioara Bintu,
Identifier: pmid:37348463
Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression. Given their multifunctional nature and high sequence divergence, it is unclear which viral proteins can affect transcription and which specific sequences contribute to this function. Using a high-throughput assay, we measured the transcriptional regulatory potential of over 60,000 protein tiles across ∼1,500 proteins from 11 coronaviruses and all nine human herpesviruses. We discovered......Read more
Large-scale map of RNA binding protein interactomes across the mRNA life-cycle
Publication: bioRxiv : the preprint server for biology
Date: Mon, 19 Jun 2023
Authors: Lena Street, Katherine Rothamel, Kristopher Brannan, Wenhao Jin, Benjamin Bokor, Kevin Dong, Kevin Rhine, Assael Madrigal, Norah Al-Azzam, Jenny Kim Kim, Yanzhe Ma, Ahmed Abdou, Erica Wolin, Ella Doron-Mandel, Joshua Ahdout, Mayuresh Mujumdar, Marko Jovanovic, Gene W Yeo,
Identifier: pmid:37333282
Messenger RNAs (mRNAs) interact with RNA-binding proteins (RBPs) in diverse ribonucleoprotein complexes (RNPs) during distinct life-cycle stages for their processing and maturation. While substantial attention has focused on understanding RNA regulation by assigning proteins, particularly RBPs, to specific RNA substrates, there has been considerably less exploration leveraging protein-protein interaction (PPI) methodologies to identify and study the role of proteins in mRNA life-cycle stages. To......Read more
SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a potential mechanism for selective translational suppression upon cellular stress
Publication: bioRxiv : the preprint server for biology
Date: Mon, 19 Jun 2023
Authors: Erica Wolin, Jimmy K Guo, Mario R Blanco, Andrew A Perez, Isabel N Goronzy, Ahmed A Abdou, Darvesh Gorhe, Mitchell Guttman, Marko Jovanovic,
Identifier: pmid:37333139
RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To......Read more
ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment
Publication: Cell reports
Date: Sun, 18 Jun 2023
Authors: Jeffrey D Steimle, Chul Kim, Megan Rowton, Rangarajan D Nadadur, Zhezhen Wang, Matthew Stocker, Andrew D Hoffmann, Erika Hanson, Junghun Kweon, Tanvi Sinha, Kyunghee Choi, Brian L Black, John M Cunningham, Ivan P Moskowitz, Kohta Ikegami,
Identifier: pmid:37330911
Mechanisms underlying distinct specification, commitment, and differentiation phases of cell fate determination remain undefined due to difficulties capturing these processes. Here, we interrogate the activity of ETV2, a transcription factor necessary and sufficient for hematoendothelial differentiation, within isolated fate intermediates. We observe transcriptional upregulation of Etv2 and opening of ETV2-binding sites, indicating new ETV2 binding, in a common cardiac-hematoendothelial......Read more
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution
Publication: Nature chemical biology
Date: Thu, 15 Jun 2023
Authors: Tong Wang, Johanna M Fowler, Laura Liu, Christian E Loo, Meiqi Luo, Emily K Schutsky, Kiara N Berríos, Jamie E DeNizio, Ashley Dvorak, Nick Downey, Saira Montermoso, Bianca Y Pingul, MacLean Nasrallah, Walraj S Gosal, Hao Wu, Rahul M Kohli,
Identifier: pmid:37322153
5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes. The ideal method for 5mC localization would be both nondestructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here we present direct methylation sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution using nanogram quantities of DNA. DM-Seq employs two key DNA-modifying enzymes: a neomorphic DNA methyltransferase and a DNA deaminase......Read more
Dual chemical labeling enables nucleotide-resolution mapping of DNA abasic sites and common alkylation damage in human mitochondrial DNA
Publication: Nucleic acids research
Date: Fri, 09 Jun 2023
Authors: Chaoxing Liu, Brandon H Le, Wenyan Xu, Ching-Hsin Yang, Yu Hsuan Chen, Linlin Zhao,
Identifier: pmid:37293974
Mitochondrial DNA (mtDNA) modifications play an emerging role in innate immunity and inflammatory diseases. Nonetheless, relatively little is known regarding the locations of mtDNA modifications. Such information is critically important for deciphering their roles in mtDNA instability, mtDNA-mediated immune and inflammatory responses, and mitochondrial disorders. The affinity probe-based enrichment of lesion-containing DNA represents a key strategy for sequencing DNA modifications. Existing......Read more
Droplet-based bisulfite sequencing for high-throughput profiling of single-cell DNA methylomes
Publication: bioRxiv : the preprint server for biology
Date: Fri, 09 Jun 2023
Authors: Qiang Zhang, Sai Ma, Zhengzhi Liu, Bohan Zhu, Zirui Zhou, Gaoshan Li, J Javier Meana, Javier González-Maeso, Chang Lu,
Identifier: pmid:37293095
Genome-wide DNA methylation profile, or DNA methylome, is a critical component of the overall epigenomic landscape that modulates gene activities and cell fate. Single-cell DNA methylomic studies offer unprecedented resolution for detecting and profiling cell subsets based on methylomic features. However, existing single-cell methylomic technologies are all based on use of tubes or well plates and these platforms are not easily scalable for handling a large number of single cells. Here we......Read more
MRT-ModSeq - Rapid detection of RNA modifications with MarathonRT
Publication: bioRxiv : the preprint server for biology
Date: Fri, 09 Jun 2023
Authors: Rafael de Cesaris Araujo Tavares, Gandhar Mahadeshwar, Han Wan, Anna Marie Pyle,
Identifier: pmid:37292902
Chemical modifications are essential regulatory elements that modulate the behavior and function of cellular RNAs. Despite recent advances in sequencing-based RNA modification mapping, methods combining accuracy and speed are still lacking. Here, we introduce MRT- ModSeq for rapid, simultaneous detection of multiple RNA modifications using MarathonRT. MRT-ModSeq employs distinct divalent cofactors to generate 2-D mutational profiles that are highly dependent on nucleotide identity and......Read more
The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity
Publication: The CRISPR journal
Date: Mon, 05 Jun 2023
Authors: Suchita P Nety, Han Altae-Tran, Soumya Kannan, F Esra Demircioglu, Guilhem Faure, Seiichi Hirano, Kepler Mears, Yugang Zhang, Rhiannon K Macrae, Feng Zhang,
Identifier: pmid:37272862
TnpB is a member of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family, is harbored in transposons, and likely functions to maintain the transposon in genomes. Previously, it was shown that TnpB cleaves double- and single-stranded DNA substrates in an RNA-guided manner, but the biogenesis of the TnpB ribonucleoprotein (RNP) complex is unknown. Using in vitro purified apo TnpB, we demonstrate the ability of TnpB to generate guide omegaRNA (ωRNA) from its own mRNA......Read more
Modularity and diversity of target selectors in Tn7 transposons
Publication: Molecular cell
Date: Fri, 02 Jun 2023
Authors: Guilhem Faure, Makoto Saito, Sean Benler, Iris Peng, Yuri I Wolf, Jonathan Strecker, Han Altae-Tran, Edwin Neumann, David Li, Kira S Makarova, Rhiannon K Macrae, Eugene V Koonin, Feng Zhang,
Identifier: pmid:37267947
To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously......Read more
Multimodal perturbation analyses of cyclin-dependent kinases reveal a network of synthetic lethalities associated with cell-cycle regulation and transcriptional regulation
Publication: Scientific reports
Date: Thu, 11 May 2023
Authors: Kyle Ford, Brenton P Munson, Samson H Fong, Rebecca Panwala, Wai Keung Chu, Joseph Rainaldi, Nongluk Plongthongkum, Vinayagam Arunachalam, Jarek Kostrowicki, Dario Meluzzi, Jason F Kreisberg, Kristen Jensen-Pergakes, Todd VanArsdale, Thomas Paul, Pablo Tamayo, Kun Zhang, Jadwiga Bienkowska, Prashant Mali, Trey Ideker,
Identifier: pmid:37169829
Cell-cycle control is accomplished by cyclin-dependent kinases (CDKs), motivating extensive research into CDK targeting small-molecule drugs as cancer therapeutics. Here we use combinatorial CRISPR/Cas9 perturbations to uncover an extensive network of functional interdependencies among CDKs and related factors, identifying 43 synthetic-lethal and 12 synergistic interactions. We dissect CDK perturbations using single-cell RNAseq, for which we develop a novel computational framework to precisely......Read more
The MMP-2 histone H3 N-terminal tail protease is selectively targeted to the transcription start sites of active genes
Publication: Epigenetics & chromatin
Date: Wed, 10 May 2023
Authors: Benjamin H Weekley, Judd C Rice,
Identifier: pmid:37161413
CONCLUSIONS: This study revealed that the MMP-2 H3NT protease is selectively targeted to the TSSs of active protein coding genes in U2OS cells. The resulting H3NT proteolysis directly alters local histone H3 PTM patterns at TSSs, which likely functions to regulate transcription. MMP-2 mediated H3NT proteolysis directly activates CTSB, a secondary H3NT protease that generates additional cleaved H3 products within chromatin....Read more
Beyond assembly: the increasing flexibility of single-molecule sequencing technology
Publication: Nature reviews. Genetics
Date: Wed, 10 May 2023
Authors: Paul W Hook, Winston Timp,
Identifier: pmid:37161088
The maturation of high-throughput short-read sequencing technology over the past two decades has shaped the way genomes are studied. Recently, single-molecule, long-read sequencing has emerged as an essential tool in deciphering genome structure and function, including filling gaps in the human reference genome, measuring the epigenome and characterizing splicing variants in the transcriptome. With recent technological developments, these single-molecule technologies have moved beyond genome......Read more
Dam Assisted Fluorescent Tagging of Chromatin Accessibility (DAFCA) for Optical Genome Mapping in Nanochannel Arrays
Publication: ACS nano
Date: Mon, 08 May 2023
Authors: Gil Nifker, Assaf Grunwald, Sapir Margalit, Zuzana Tulpova, Yael Michaeli, Hagai Har-Gil, Noy Maimon, Elad Roichman, Leonie Schütz, Elmar Weinhold, Yuval Ebenstein,
Identifier: pmid:37154345
Proteins and enzymes in the cell nucleus require physical access to their DNA target sites in order to perform genomic tasks such as gene activation and transcription. Hence, chromatin accessibility is a central regulator of gene expression, and its genomic profile holds essential information on the cell type and state. We utilized the E. coli Dam methyltransferase in combination with a fluorescent cofactor analogue to generate fluorescent tags in accessible DNA regions within the cell nucleus.......Read more
Substantial Slowing of Electrophoretic Translocation of DNA through a Nanopore Using Coherent Multiple Entropic Traps
Publication: ACS nano
Date: Fri, 05 May 2023
Authors: Kuo Chen, Murugappan Muthukumar,
Identifier: pmid:37146154
One of the major challenges in the technology of sequencing DNA using single-molecule electrophoresis through a nanopore is to control the translocation of the macromolecule across the pore in order to allow sufficient time for accurate sequence reading at limited recording bandwidths. If the translocation speed is too fast, the signatures of the bases passing through the sensing region of the nanopore overlap in time, presenting difficulties in accurately identifying the bases in a sequential......Read more
Photoselective sequencing: microscopically guided genomic measurements with subcellular resolution
Publication: Nature methods
Date: Thu, 27 Apr 2023
Authors: Sarah M Mangiameli, Haiqi Chen, Andrew S Earl, Julie A Dobkin, Daniel Lesman, Jason D Buenrostro, Fei Chen,
Identifier: pmid:37106232
In biological systems, spatial organization and function are interconnected. Here we present photoselective sequencing, a new method for genomic and epigenomic profiling within morphologically distinct regions. Starting with an intact biological specimen, photoselective sequencing uses targeted illumination to selectively unblock a photocaged fragment library, restricting the sequencing-based readout to microscopically identified spatial regions. We validate photoselective sequencing by......Read more
Promoter-independent synthesis of chemically modified RNA by human DNA polymerase θ variants
Publication: RNA (New York, N.Y.)
Date: Thu, 27 Apr 2023
Authors: Taylor Tredinnick, Tatiana Kent, Leonid Minakhin, Ziyuan Li, Jozef Madzo, Xiaojiang S Chen, Richard T Pomerantz,
Identifier: pmid:37105714
Synthetic RNA oligonucleotides composed of canonical and modified ribonucleotides are highly effective for RNA antisense therapeutics and RNA-based genome engineering applications utilizing CRISPR-Cas9. Yet, synthesis of synthetic RNA using phosphoramidite chemistry is highly inefficient and expensive relative to DNA oligonucleotides, especially for relatively long RNA oligonucleotides. Thus, new biotechnologies are needed to significantly reduce costs, while increasing synthesis rates and......Read more
Multicellular, IVT-derived, unmodified human transcriptome for nanopore direct RNA analysis
Publication: bioRxiv : the preprint server for biology
Date: Mon, 17 Apr 2023
Authors: Caroline A McCormick, Stuart Akeson, Sepideh Tavakoli, Dylan Bloch, Isabel N Klink, Miten Jain, Sara H Rouhanifard,
Identifier: pmid:37066160
Nanopore direct RNA sequencing (DRS) enables measurements of native RNA modifications. Modification-free transcripts are an important control for DRS. Additionally, it is advantageous to have canonical transcripts from multiple cell lines to better account for human transcriptome variation. Here we generated and analyzed Nanopore DRS datasets for five human cell lines using in vitro transcribed (IVT) RNA. We compared performance statistics amongst biological replicates. We also documented......Read more
A genome-wide optical pooled screen reveals regulators of cellular antiviral responses
Publication: Proceedings of the National Academy of Sciences of the United States of America
Date: Wed, 12 Apr 2023
Authors: Rebecca J Carlson, Michael D Leiken, Alina Guna, Nir Hacohen, Paul C Blainey,
Identifier: pmid:37043539
The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins. Here, we increased the throughput of our......Read more
Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription
Publication: Science (New York, N.Y.)
Date: Thu, 06 Apr 2023
Authors: Max E Wilkinson, Chris J Frangieh, Rhiannon K Macrae, Feng Zhang,
Identifier: pmid:37023171
Non-long terminal repeat (non-LTR) retrotransposons, or long interspersed nuclear elements (LINEs), are an abundant class of eukaryotic transposons that insert into genomes by target-primed reverse transcription (TPRT). During TPRT, a target DNA sequence is nicked and primes reverse transcription of the retrotransposon RNA. Here, we report the cryo-electron microscopy structure of the Bombyx mori R2 non-LTR retrotransposon initiating TPRT at its ribosomal DNA target. The target DNA sequence is......Read more
Cryo-EM structure of the transposon-associated TnpB enzyme
Publication: Nature
Date: Wed, 05 Apr 2023
Authors: Ryoya Nakagawa, Hisato Hirano, Satoshi N Omura, Suchita Nety, Soumya Kannan, Han Altae-Tran, Xiao Yao, Yuriko Sakaguchi, Takayuki Ohira, Wen Y Wu, Hiroshi Nakayama, Yutaro Shuto, Tatsuki Tanaka, Fumiya K Sano, Tsukasa Kusakizako, Yoshiaki Kise, Yuzuru Itoh, Naoshi Dohmae, John van der Oost, Tsutomu Suzuki, Feng Zhang, Osamu Nureki,
Identifier: pmid:37020030
The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins¹. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases^(2,3). TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the......Read more
Large-scale mapping and mutagenesis of human transcriptional effector domains
Publication: Nature
Date: Wed, 05 Apr 2023
Authors: Nicole DelRosso, Josh Tycko, Peter Suzuki, Cecelia Andrews, None Aradhana, Adi Mukund, Ivan Liongson, Connor Ludwig, Kaitlyn Spees, Polly Fordyce, Michael C Bassik, Lacramioara Bintu,
Identifier: pmid:37020022
Human gene expression is regulated by more than 2,000 transcription factors and chromatin regulators^(1,2). Effector domains within these proteins can activate or repress transcription. However, for many of these regulators we do not know what type of effector domains they contain, their location in the protein, their activation and repression strengths, and the sequences that are necessary for their functions. Here, we systematically measure the effector activity of more than 100,000 protein......Read more
mRNA Regulation by RNA Modifications
Publication: Annual review of biochemistry
Date: Wed, 05 Apr 2023
Authors: Wendy V Gilbert, Sigrid Nachtergaele,
Identifier: pmid:37018844
Chemical modifications on mRNA represent a critical layer of gene expression regulation. Research in this area has continued to accelerate over the last decade, as more modifications are being characterized with increasing depth and breadth. mRNA modifications have been demonstrated to influence nearly every step from the early phases of transcript synthesis in the nucleus through to their decay in the cytoplasm, but in many cases, the molecular mechanisms involved in these processes remain......Read more
Programmable protein delivery with a bacterial contractile injection system
Publication: Nature
Date: Wed, 29 Mar 2023
Authors: Joseph Kreitz, Mirco J Friedrich, Akash Guru, Blake Lash, Makoto Saito, Rhiannon K Macrae, Feng Zhang,
Identifier: pmid:36991127
Endosymbiotic bacteria have evolved intricate delivery systems that enable these organisms to interface with host biology. One example, the extracellular contractile injection systems (eCISs), are syringe-like macromolecular complexes that inject protein payloads into eukaryotic cells by driving a spike through the cellular membrane. Recently, eCISs have been found to target mouse cells^(1-3), raising the possibility that these systems could be harnessed for therapeutic protein delivery.......Read more
Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases
Publication: Nature biotechnology
Date: Wed, 29 Mar 2023
Authors: George D Lampe, Rebeca T King, Tyler S Halpin-Healy, Sanne E Klompe, Marcus I Hogan, Phuc Leo H Vo, Stephen Tang, Alejandro Chavez, Samuel H Sternberg,
Identifier: pmid:36991112
Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment......Read more
Precision engineering of biological function with large-scale measurements and machine learning
Publication: PloS one
Date: Wed, 29 Mar 2023
Authors: Drew S Tack, Peter D Tonner, Abe Pressman, Nathan D Olson, Sasha F Levy, Eugenia F Romantseva, Nina Alperovich, Olga Vasilyeva, David Ross,
Identifier: pmid:36989327
As synthetic biology expands and accelerates into real-world applications, methods for quantitatively and precisely engineering biological function become increasingly relevant. This is particularly true for applications that require programmed sensing to dynamically regulate gene expression in response to stimuli. However, few methods have been described that can engineer biological sensing with any level of quantitative precision. Here, we present two complementary methods for precision......Read more
RNA Polymerase II pausing temporally coordinates cell cycle progression and erythroid differentiation
Publication: medRxiv : the preprint server for health sciences
Date: Wed, 22 Mar 2023
Authors: Danya J Martell, Hope E Merens, Claudia Fiorini, Alexis Caulier, Jacob C Ulirsch, Robert Ietswaart, Karine Choquet, Giovanna Graziadei, Valentina Brancaleoni, Maria Domenica Cappellini, Caroline Scott, Nigel Roberts, Melanie Proven, Noémi Ba Roy, Christian Babbs, Douglas R Higgs, Vijay G Sankaran, L Stirling Churchman,
Identifier: pmid:36945604
The controlled release of promoter-proximal paused RNA polymerase II (Pol II) into productive elongation is a major step in gene regulation. However, functional analysis of Pol II pausing is difficult because factors that regulate pause release are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H , which encodes SPT5, in individuals with β-thalassemia unlinked to HBB mutations. During erythropoiesis in healthy human cells, cell cycle genes were......Read more
High-Capacity Sample Multiplexing for Single Cell Chromatin Accessibility Profiling
Publication: bioRxiv : the preprint server for biology
Date: Wed, 22 Mar 2023
Authors: Gregory T Booth, Riza M Daza, Sanjay R Srivatsan, Jos L McFaline-Figueroa, Rula Green Gladden, Scott N Furlan, Jay Shendure, Cole Trapnell,
Identifier: pmid:36945538
Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first......Read more
Tigerfish designs oligonucleotide-based in situ hybridization probes targeting intervals of highly repetitive DNA at the scale of genomes
Publication: bioRxiv : the preprint server for biology
Date: Wed, 22 Mar 2023
Authors: Robin Aguilar, Conor K Camplisson, Qiaoyi Lin, Karen H Miga, William S Noble, Brian J Beliveau,
Identifier: pmid:36945528
Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate >100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific......Read more
An integrated platform for high-throughput nanoscopy
Publication: Nature biotechnology
Date: Tue, 14 Mar 2023
Authors: Andrew E S Barentine, Yu Lin, Edward M Courvan, Phylicia Kidd, Miao Liu, Leonhard Balduf, Timy Phan, Felix Rivera-Molina, Michael R Grace, Zach Marin, Mark Lessard, Juliana Rios Chen, Siyuan Wang, Karla M Neugebauer, Joerg Bewersdorf, David Baddeley,
Identifier: pmid:36914886
Single-molecule localization microscopy enables three-dimensional fluorescence imaging at tens-of-nanometer resolution, but requires many camera frames to reconstruct a super-resolved image. This limits the typical throughput to tens of cells per day. While frame rates can now be increased by over an order of magnitude, the large data volumes become limiting in existing workflows. Here we present an integrated acquisition and analysis platform leveraging microscopy-specific data compression,......Read more
Sampling the proteome by emerging single-molecule and mass spectrometry methods
Publication: Nature methods
Date: Fri, 10 Mar 2023
Authors: Michael J MacCoss, Javier Antonio Alfaro, Danielle A Faivre, Christine C Wu, Meni Wanunu, Nikolai Slavov,
Identifier: pmid:36899164
Mammalian cells have about 30,000-fold more protein molecules than mRNA molecules, which has major implications in the development of proteomics technologies. We review strategies that have been helpful for counting billions of protein molecules by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and suggest that these strategies can benefit single-molecule methods, especially in mitigating the challenges of the wide dynamic range of the proteome....Read more
Mapping the Chemical Space of Active-Site Targeted Covalent Ligands for Protein Tyrosine Phosphatases
Publication: Chembiochem : a European journal of chemical biology
Date: Thu, 09 Mar 2023
Authors: Suk Ho Hong, Sarah Y Xi, Andrew C Johns, Lauren C Tang, Allyson Li, Madeleine N Hum, Cassandra A Chartier, Marko Jovanovic, Neel H Shah,
Identifier: pmid:36893077
Protein tyrosine phosphatases (PTPs) are an important class of enzymes that modulate essential cellular processes through protein dephosphorylation and are dysregulated in various disease states. There is demand for new compounds that target the active sites of these enzymes, for use as chemical tools to dissect their biological roles or as leads for the development of new therapeutics. In this study, we explore an array of electrophiles and fragment scaffolds to investigate the required......Read more
Phased nanopore assembly with Shasta and modular graph phasing with GFAse
Publication: bioRxiv : the preprint server for biology
Date: Fri, 03 Mar 2023
Authors: Ryan Lorig-Roach, Melissa Meredith, Jean Monlong, Miten Jain, Hugh Olsen, Brandy McNulty, David Porubsky, Tessa Montague, Julian Lucas, Chris Condon, Jordan Eizenga, Sissel Juul, Sean McKenzie, Sara E Simmonds, Jimin Park, Mobin Asri, Sergey Koren, Evan Eichler, Richard Axel, Bruce Martin, Paolo Carnevali, Karen Miga, Benedict Paten,
Identifier: pmid:36865218
As a step towards simplifying and reducing the cost of haplotype resolved de novo assembly, we describe new methods for accurately phasing nanopore data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of Oxford Nanopore Technologies’ (ONT) PromethION sequencing, including those using proximity ligation and show that newer, higher accuracy ONT reads substantially improve assembly quality....Read more
RSCanner: rapid assessment and visualization of RNA structure content
Publication: Bioinformatics (Oxford, England)
Date: Wed, 01 Mar 2023
Authors: Gandhar Mahadeshwar, Rafael de Cesaris Araujo Tavares, Han Wan, Zion R Perry, Anna Marie Pyle,
Identifier: pmid:36857576
MOTIVATION: The increasing availability of RNA structural information that spans many kilobases of transcript sequence imposes a need for tools that can rapidly screen, identify, and prioritize structural modules of interest....Read more
Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1
Publication: Biomolecules
Date: Sat, 25 Feb 2023
Authors: Harutyun Khachatryan, Bartlomiej Olszowy, Carlos A Barrero, John Gordon, Oscar Perez-Leal,
Identifier: pmid:36830618
Tubulin is a protein that plays a critical role in maintaining cellular structure and facilitating cell division. Inhibiting tubulin polymerization has been shown to be an effective strategy for inhibiting the proliferation of cancer cells. In the past, identifying compounds that could inhibit tubulin polymerization has required the use of in vitro assays utilizing purified tubulin or immunofluorescence of fixed cells. This study presents a novel approach for identifying tubulin polymerization......Read more
A nucleolar long "non-coding" RNA encodes a novel protein that functions in response to stress
Publication: Proceedings of the National Academy of Sciences of the United States of America
Date: Wed, 22 Feb 2023
Authors: Shuang Feng, Anthony Desotell, Alison Ross, Marko Jovanovic, James L Manley,
Identifier: pmid:36812203
Certain long non-coding RNAs (lncRNAs) are known to contain small open reading frames that can be translated. Here we describe a much larger 25 kDa human protein, "Ribosomal IGS Encoded Protein" (RIEP), that remarkably is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar "promoter and pre-rRNA antisense" lncRNA (PAPAS). Strikingly, RIEP, which is conserved throughout primates but not found in other species, predominantly localizes to the nucleolus as well as......Read more
Alternative polyadenylation alters protein dosage by switching between intronic and 3'UTR sites
Publication: Science advances
Date: Fri, 17 Feb 2023
Authors: Nicola de Prisco, Caitlin Ford, Nathan D Elrod, Winston Lee, Lauren C Tang, Kai-Lieh Huang, Ai Lin, Ping Ji, Venkata S Jonnakuti, Lia Boyle, Maximilian Cabaj, Salvatore Botta, Katrin Õunap, Karit Reinson, Monica H Wojcik, Jill A Rosenfeld, Weimin Bi, Kristian Tveten, Trine Prescott, Thorsten Gerstner, Audrey Schroeder, Chin-To Fong, Jaya K George-Abraham, Catherine A Buchanan, Andrea Hanson-Khan, Jonathan A Bernstein, Aikaterini A Nella, Wendy K Chung, Vicky Brandt, Marko Jovanovic, Kimara L Targoff, Hari Krishna Yalamanchili, Eric J Wagner, Vincenzo A Gennarino,
Identifier: pmid:36800428
Alternative polyadenylation (APA) creates distinct transcripts from the same gene by cleaving the pre-mRNA at poly(A) sites that can lie within the 3' untranslated region (3'UTR), introns, or exons. Most studies focus on APA within the 3'UTR; however, here, we show that CPSF6 insufficiency alters protein levels and causes a developmental syndrome by deregulating APA throughout the transcript. In neonatal humans and zebrafish larvae, CPSF6 insufficiency shifts poly(A) site usage between the 3'UTR......Read more
The multi-tissue landscape of somatic mtDNA mutations indicates tissue-specific accumulation and removal in aging
Publication: eLife
Date: Fri, 17 Feb 2023
Authors: Monica Sanchez-Contreras, Mariya T Sweetwyne, Kristine A Tsantilas, Jeremy A Whitson, Matthew D Campbell, Brenden F Kohrn, Hyeon Jeong Kim, Michael J Hipp, Jeanne Fredrickson, Megan M Nguyen, James B Hurley, David J Marcinek, Peter S Rabinovitch, Scott R Kennedy,
Identifier: pmid:36799304
Accumulation of somatic mutations in the mitochondrial genome (mtDNA) has long been proposed as a possible mechanism of mitochondrial and tissue dysfunction that occurs during aging. A thorough characterization of age-associated mtDNA somatic mutations has been hampered by the limited ability to detect low-frequency mutations. Here, we used Duplex Sequencing on eight tissues of an aged mouse cohort to detect >89,000 independent somatic mtDNA mutations and show significant tissue-specific......Read more
SEC-TMT facilitates quantitative differential analysis of protein interaction networks
Publication: bioRxiv : the preprint server for biology
Date: Mon, 30 Jan 2023
Authors: Ella Doron-Mandel, Benjamin J Bokor, Yanzhe Ma, Lena A Street, Lauren C Tang, Ahmed A Abdou, Neel H Shah, George A Rosenberger, Marko Jovanovic,
Identifier: pmid:36711903
The majority of cellular proteins interact with at least one partner or assemble into molecular-complexes to exert their function. This network of protein-protein interactions (PPIs) and the composition of macromolecular machines differ between cell types and physiological conditions. Therefore, characterizing PPI networks and their dynamic changes is vital for discovering novel biological functions and underlying mechanisms of cellular processes. However, producing an in-depth, global snapshot......Read more
Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation
Publication: bioRxiv : the preprint server for biology
Date: Mon, 30 Jan 2023
Authors: Mikhail Kolmogorov, Kimberley J Billingsley, Mira Mastoras, Melissa Meredith, Jean Monlong, Ryan Lorig-Roach, Mobin Asri, Pilar Alvarez Jerez, Laksh Malik, Ramita Dewan, Xylena Reed, Rylee M Genner, Kensuke Daida, Sairam Behera, Kishwar Shafin, Trevor Pesout, Jeshuwin Prabakaran, Paolo Carnevali, North American Brain Expression Consortium (NABEC), Jianzhi Yang, Arang Rhie, Sonja W Scholz, Bryan J Traynor, Karen H Miga, Miten Jain, Winston Timp, Adam M Phillippy, Mark Chaisson, Fritz J Sedlazeck, Cornelis Blauwendraat, Benedict Paten,
Identifier: pmid:36711673
Long-read sequencing technologies substantially overcome the limitations of short-reads but to date have not been considered as feasible replacement at scale due to a combination of being too expensive, not scalable enough, or too error-prone. Here, we develop an efficient and scalable wet lab and computational protocol for Oxford Nanopore Technologies (ONT) long-read sequencing that seeks to provide a genuine alternative to short-reads for large-scale genomics projects. We applied our protocol......Read more
Parent tRNA Modification Status Determines the Induction of Functional tRNA-Derived RNA by Respiratory Syncytial Virus Infection
Publication: Viruses
Date: Sat, 21 Jan 2023
Authors: Eun-Jin Choi, Wenzhe Wu, Ke Zhang, Xiaohong Yuan, Junfang Deng, Deena Ismail, Darby L Buck, Kerrie S Thomason, Roberto P Garofalo, Shenglong Zhang, Xiaoyong Bao,
Identifier: pmid:36680097
tRNA-derived RNA fragments (tRFs) are a recently discovered family of small noncoding RNAs (sncRNAs). We previously reported that respiratory syncytial virus (RSV) infection induces functional tRFs, which are derived from a limited subset of parent tRNAs, in airway epithelial cells. Such induction is also observed in nasopharyngeal wash samples from RSV patients and correlates to RSV genome copies, suggesting a clinical significance of tRFs in RSV infection. This work also investigates whether......Read more
Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing
Publication: Nature communications
Date: Thu, 19 Jan 2023
Authors: Sepideh Tavakoli, Mohammad Nabizadeh, Amr Makhamreh, Howard Gamper, Caroline A McCormick, Neda K Rezapour, Ya-Ming Hou, Meni Wanunu, Sara H Rouhanifard,
Identifier: pmid:36658122
Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover......Read more
Navigating the pitfalls of mapping DNA and RNA modifications
Publication: Nature reviews. Genetics
Date: Wed, 18 Jan 2023
Authors: Yimeng Kong, Edward A Mead, Gang Fang,
Identifier: pmid:36653550
Chemical modifications to nucleic acids occur across the kingdoms of life and carry important regulatory information. Reliable high-resolution mapping of these modifications is the foundation of functional and mechanistic studies, and recent methodological advances based on next-generation sequencing and long-read sequencing platforms are critical to achieving this aim. However, mapping technologies may have limitations that sometimes lead to inconsistent results. Some of these limitations are......Read more
Comprehensive variant discovery in the era of complete human reference genomes
Publication: Nature methods
Date: Thu, 12 Jan 2023
Authors: Monika Cechova, Karen H Miga,
Identifier: pmid:36635553
Advances in long-read sequencing technologies have broadened our understanding of genetic variation in the human population, uncovered new complex structural variants and offered an opportunity to elucidate new variant associations with disease....Read more
A single-cell massively parallel reporter assay detects cell-type-specific gene regulation
Publication: Nature genetics
Date: Thu, 12 Jan 2023
Authors: Siqi Zhao, Clarice K Y Hong, Connie A Myers, David M Granas, Michael A White, Joseph C Corbo, Barak A Cohen,
Identifier: pmid:36635387
Massively parallel reporter gene assays are key tools in regulatory genomics but cannot be used to identify cell-type-specific regulatory elements without performing assays serially across different cell types. To address this problem, we developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell types simultaneously. We assayed a library of core promoters in a mixture of HEK293 and K562 cells......Read more
Unidirectional single-file transport of full-length proteins through a nanopore
Publication: Nature biotechnology
Date: Mon, 09 Jan 2023
Authors: Luning Yu, Xinqi Kang, Fanjun Li, Behzad Mehrafrooz, Amr Makhamreh, Ali Fallahi, Joshua C Foster, Aleksei Aksimentiev, Min Chen, Meni Wanunu,
Identifier: pmid:36624148
The electrical current blockade of a peptide or protein threading through a nanopore can be used as a fingerprint of the molecule in biosensor applications. However, threading of full-length proteins has only been achieved using enzymatic unfolding and translocation. Here we describe an enzyme-free approach for unidirectional, slow transport of full-length proteins through nanopores. We show that the combination of a chemically resistant biological nanopore, α-hemolysin (narrowest part is ~1.4......Read more
A transcription factor atlas of directed differentiation
Publication: Cell
Date: Sat, 07 Jan 2023
Authors: Julia Joung, Sai Ma, Tristan Tay, Kathryn R Geiger-Schuller, Paul C Kirchgatterer, Vanessa K Verdine, Baolin Guo, Mario A Arias-Garcia, William E Allen, Ankita Singh, Olena Kuksenko, Omar O Abudayyeh, Jonathan S Gootenberg, Zhanyan Fu, Rhiannon K Macrae, Jason D Buenrostro, Aviv Regev, Feng Zhang,
Identifier: pmid:36608654
Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated......Read more
Single-cell transcriptomic profiling of the zebrafish inner ear reveals molecularly distinct hair cell and supporting cell subtypes
Publication: eLife
Date: Wed, 04 Jan 2023
Authors: Tuo Shi, Marielle O Beaulieu, Lauren M Saunders, Peter Fabian, Cole Trapnell, Neil Segil, J Gage Crump, David W Raible,
Identifier: pmid:36598134
A major cause of human deafness and vestibular dysfunction is permanent loss of the mechanosensory hair cells of the inner ear. In non-mammalian vertebrates such as zebrafish, regeneration of missing hair cells can occur throughout life. While a comparative approach has the potential to reveal the basis of such differential regenerative ability, the degree to which the inner ears of fish and mammals share common hair cells and supporting cell types remains unresolved. Here, we perform......Read more
Mammalian DNA methylome dynamics: mechanisms, functions and new frontiers
Publication: Development (Cambridge, England)
Date: Thu, 15 Dec 2022
Authors: Alex Wei, Hao Wu,
Identifier: pmid:36519514
DNA methylation is a highly conserved epigenetic modification that plays essential roles in mammalian gene regulation, genome stability and development. Despite being primarily considered a stable and heritable epigenetic silencing mechanism at heterochromatic and repetitive regions, whole genome methylome analysis reveals that DNA methylation can be highly cell-type specific and dynamic within proximal and distal gene regulatory elements during early embryonic development, stem cell......Read more
Rapid Identification of DNA Fragments through Direct Sequencing with Electro-Optical Zero-Mode Waveguides
Publication: Advanced materials (Deerfield Beach, Fla.)
Date: Fri, 09 Dec 2022
Authors: Fatemeh Farhangdoust, Mohammad Amin Alibakhshi, Feng Cheng, Wentao Liang, Yongmin Liu, Meni Wanunu,
Identifier: pmid:36482018
No abstract...Read more
Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases
Publication: Nature biotechnology
Date: Thu, 24 Nov 2022
Authors: Matthew T N Yarnall, Eleonora I Ioannidi, Cian Schmitt-Ulms, Rohan N Krajeski, Justin Lim, Lukas Villiger, Wenyuan Zhou, Kaiyi Jiang, Sofya K Garushyants, Nathaniel Roberts, Liyang Zhang, Christopher A Vakulskas, John A Walker, Anastasia P Kadina, Adrianna E Zepeda, Kevin Holden, Hong Ma, Jun Xie, Guangping Gao, Lander Foquet, Greg Bial, Sara K Donnelly, Yoshinari Miyata, Daniel R Radiloff, Jordana M Henderson, Andrew Ujita, Omar O Abudayyeh, Jonathan S Gootenberg,
Identifier: pmid:36424489
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci......Read more
Quantitative fate mapping: A general framework for analyzing progenitor state dynamics via retrospective lineage barcoding
Publication: Cell
Date: Thu, 24 Nov 2022
Authors: Weixiang Fang, Claire M Bell, Abel Sapirstein, Soichiro Asami, Kathleen Leeper, Donald J Zack, Hongkai Ji, Reza Kalhor,
Identifier: pmid:36423582
Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct......Read more
RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease
Publication: Science (New York, N.Y.)
Date: Thu, 24 Nov 2022
Authors: Kazuki Kato, Sae Okazaki, Cian Schmitt-Ulms, Kaiyi Jiang, Wenyuan Zhou, Junichiro Ishikawa, Yukari Isayama, Shungo Adachi, Tomohiro Nishizawa, Kira S Makarova, Eugene V Koonin, Omar O Abudayyeh, Jonathan S Gootenberg, Hiroshi Nishimasu,
Identifier: pmid:36423304
The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage......Read more
RNA-activated protein cleavage with a CRISPR-associated endopeptidase
Publication: Science (New York, N.Y.)
Date: Thu, 24 Nov 2022
Authors: Jonathan Strecker, F Esra Demircioglu, David Li, Guilhem Faure, Max E Wilkinson, Jonathan S Gootenberg, Omar O Abudayyeh, Hiroshi Nishimasu, Rhiannon K Macrae, Feng Zhang,
Identifier: pmid:36423276
In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this......Read more
CD3e-immunotoxin spares CD62Llo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehi T cells
Publication: Frontiers in immunology
Date: Thu, 17 Nov 2022
Authors: Shihyoung Kim, Rajni Kant Shukla, Hannah Yu, Alice Baek, Sophie G Cressman, Sarah Golconda, Ga-Eun Lee, Hyewon Choi, John C Reneau, Zhirui Wang, Christene A Huang, Namal P M Liyanage, Sanggu Kim,
Identifier: pmid:36389741
CD3-epsilon(CD3e) immunotoxins (IT), a promising precision reagent for various clinical conditions requiring effective depletion of T cells, often shows limited treatment efficacy for largely unknown reasons. Tissue-resident T cells that persist in peripheral tissues have been shown to play pivotal roles in local and systemic immunity, as well as transplant rejection, autoimmunity and cancers. The impact of CD3e-IT treatment on these local cells, however, remains poorly understood. Here, using a......Read more
Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity
Publication: The CRISPR journal
Date: Tue, 15 Nov 2022
Authors: Alyssa Hoy, Ya Ying Zheng, Jia Sheng, Maksim Royzen,
Identifier: pmid:36378256
The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the incorporation of modifications into the single-guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs......Read more
UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay
Publication: Nucleic acids research
Date: Sat, 12 Nov 2022
Authors: Joseph H Chapman, Jonathan M Craig, Clara D Wang, Jens H Gundlach, Keir C Neuman, J Robert Hogg,
Identifier: pmid:36370101
The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities,......Read more
Pooled genetic screens with image-based profiling
Publication: Molecular systems biology
Date: Fri, 11 Nov 2022
Authors: Russell T Walton, Avtar Singh, Paul C Blainey,
Identifier: pmid:36366905
Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to......Read more
The phenotypic landscape of essential human genes
Publication: Cell
Date: Tue, 08 Nov 2022
Authors: Luke Funk, Kuan-Chung Su, Jimmy Ly, David Feldman, Avtar Singh, Brittania Moodie, Paul C Blainey, Iain M Cheeseman,
Identifier: pmid:36347254
Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified......Read more
Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution
Publication: Nature biotechnology
Date: Thu, 27 Oct 2022
Authors: Qing Dai, Li-Sheng Zhang, Hui-Lung Sun, Kinga Pajdzik, Lei Yang, Chang Ye, Cheng-Wei Ju, Shun Liu, Yuru Wang, Zhong Zheng, Linda Zhang, Bryan T Harada, Xiaoyang Dou, Iryna Irkliyenko, Xinran Feng, Wen Zhang, Tao Pan, Chuan He,
Identifier: pmid:36302989
Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different......Read more
Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR
Publication: Nature biotechnology
Date: Thu, 27 Oct 2022
Authors: Kaiyi Jiang, Jeremy Koob, Xi Dawn Chen, Rohan N Krajeski, Yifan Zhang, Verena Volf, Wenyuan Zhou, Samantha R Sgrizzi, Lukas Villiger, Jonathan S Gootenberg, Fei Chen, Omar O Abudayyeh,
Identifier: pmid:36302988
Programmable approaches to sense and respond to the presence of specific RNAs in biological systems have broad applications in research, diagnostics, and therapeutics. Here we engineer a programmable RNA-sensing technology, reprogrammable ADAR sensors (RADARS), which harnesses RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a cargo protein by the presence of endogenous RNA transcripts. Introduction of a stop codon in a guide upstream of the cargo makes translation......Read more
Optimized single-nucleus transcriptional profiling by combinatorial indexing
Publication: Nature protocols
Date: Wed, 19 Oct 2022
Authors: Beth K Martin, Chengxiang Qiu, Eva Nichols, Melissa Phung, Rula Green-Gladden, Sanjay Srivatsan, Ronnie Blecher-Gonen, Brian J Beliveau, Cole Trapnell, Junyue Cao, Jay Shendure,
Identifier: pmid:36261634
Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here, we report a simplified, optimized version of the sci-RNA-seq protocol with three rounds of split-pool indexing that is faster, more......Read more
Inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution
Publication: bioRxiv : the preprint server for biology
Date: Fri, 14 Oct 2022
Authors: Sinduja K Marx, Keith J Mickolajczyk, Jonathan M Craig, Christopher A Thomas, Akira M Pfeffer, Sarah J Abell, Jessica D Carrasco, Michaela C Franzi, Jesse R Huang, Hwanhee C Kim, Henry D Brinkerhoff, Tarun M Kapoor, Jens H Gundlach, Andrew H Laszlo,
Identifier: pmid:36238723
The genome of SARS-CoV-2 encodes for a helicase called nsp13 that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds short DNA duplexes. Our data confirm that nsp13......Read more
Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA
Publication: Nature
Date: Wed, 12 Oct 2022
Authors: Seiichi Hirano, Kalli Kappel, Han Altae-Tran, Guilhem Faure, Max E Wilkinson, Soumya Kannan, F Esra Demircioglu, Rui Yan, Momoko Shiozaki, Zhiheng Yu, Kira S Makarova, Eugene V Koonin, Rhiannon K Macrae, Feng Zhang,
Identifier: pmid:36224386
RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies^(1,2). Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development³. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely......Read more
Spatial maps of T cell receptors and transcriptomes reveal distinct immune niches and interactions in the adaptive immune response
Publication: Immunity
Date: Wed, 12 Oct 2022
Authors: Sophia Liu, J Bryan Iorgulescu, Shuqiang Li, Mehdi Borji, Irving A Barrera-Lopez, Vignesh Shanmugam, Haoxiang Lyu, Julia W Morriss, Zoe N Garcia, Evan Murray, David A Reardon, Charles H Yoon, David A Braun, Kenneth J Livak, Catherine J Wu, Fei Chen,
Identifier: pmid:36223726
T cells mediate antigen-specific immune responses to disease through the specificity and diversity of their clonotypic T cell receptors (TCRs). Determining the spatial distributions of T cell clonotypes in tissues is essential to understanding T cell behavior, but spatial sequencing methods remain unable to profile the TCR repertoire. Here, we developed Slide-TCR-seq, a 10-μm-resolution method, to sequence whole transcriptomes and TCRs within intact tissues. We confirmed the ability of......Read more
Microfluidic Enrichment and Computational Analysis of Rare Sequences from Mixed Genomic Samples for Metagenomic Mining
Publication: The CRISPR journal
Date: Fri, 07 Oct 2022
Authors: Naiwen Cui, Guihem Faure, Ankita Singh, Rhiannon Macrae, Feng Zhang,
Identifier: pmid:36206017
Many powerful molecular biology tools have their origins in natural systems, including restriction modification enzymes and the CRISPR effectors, Cas9, Cas12, and Cas13. Heightened interest in these systems has led to mining of genomic and metagenomic data to identify new orthologs of these proteins, new types of CRISPR systems, and uncharacterized natural systems with novel mechanisms. To accelerate metagenomic mining, we developed a high-throughput, low-cost droplet microfluidic-based method......Read more
The expanding vistas of spatial transcriptomics
Publication: Nature biotechnology
Date: Mon, 03 Oct 2022
Authors: Luyi Tian, Fei Chen, Evan Z Macosko,
Identifier: pmid:36192637
The formation and maintenance of tissue integrity requires complex, coordinated activities by thousands of genes and their encoded products. Until recently, transcript levels could only be quantified for a few genes in tissues, but advances in DNA sequencing, oligonucleotide synthesis and fluorescence microscopy have enabled the invention of a suite of spatial transcriptomics technologies capable of measuring the expression of many, or all, genes in situ. These technologies have evolved rapidly......Read more
Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion
Publication: Nucleic acids research
Date: Tue, 27 Sep 2022
Authors: Jonathan M Craig, Maria Mills, Hwanhee C Kim, Jesse R Huang, Sarah J Abell, Jonathan W Mount, Jens H Gundlach, Keir C Neuman, Andrew H Laszlo,
Identifier: pmid:36165957
Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of......Read more
Mostly natural sequencing-by-synthesis for scRNA-seq using Ultima sequencing
Publication: Nature biotechnology
Date: Thu, 15 Sep 2022
Authors: Sean K Simmons, Gila Lithwick-Yanai, Xian Adiconis, Florian Oberstrass, Nika Iremadze, Kathryn Geiger-Schuller, Pratiksha I Thakore, Chris J Frangieh, Omer Barad, Gilad Almogy, Orit Rozenblatt-Rosen, Aviv Regev, Doron Lipson, Joshua Z Levin,
Identifier: pmid:36109685
Here we introduce a mostly natural sequencing-by-synthesis (mnSBS) method for single-cell RNA sequencing (scRNA-seq), adapted to the Ultima genomics platform, and systematically benchmark it against current scRNA-seq technology. mnSBS uses mostly natural, unmodified nucleotides and only a low fraction of fluorescently labeled nucleotides, which allows for high polymerase processivity and lower costs. We demonstrate successful application in four scRNA-seq case studies of different technical and......Read more
Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs
Publication: mBio
Date: Wed, 07 Sep 2022
Authors: Jane X Yeh, Yunfan Fan, Maggie L Bartlett, Xiaoyan Zhang, Norah Sadowski, Debra A Hauer, Winston Timp, Diane E Griffin,
Identifier: pmid:36069441
Alphaviruses are positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV), the prototype alphavirus, preferentially infects neurons in mice and is a model system for studying mechanisms of viral clearance from the nervous system. Antibody specific to the SINV E2 glycoprotein plays an important role in SINV clearance, and this effect is reproduced in cultures of infected mature neurons. To determine how anti-E2 antibody affects SINV RNA synthesis,......Read more
Cell type-specific inference of differential expression in spatial transcriptomics
Publication: Nature methods
Date: Thu, 01 Sep 2022
Authors: Dylan M Cable, Evan Murray, Vignesh Shanmugam, Simon Zhang, Luli S Zou, Michael Diao, Haiqi Chen, Evan Z Macosko, Rafael A Irizarry, Fei Chen,
Identifier: pmid:36050488
A central problem in spatial transcriptomics is detecting differentially expressed (DE) genes within cell types across tissue context. Challenges to learning DE include changing cell type composition across space and measurement pixels detecting transcripts from multiple cell types. Here, we introduce a statistical method, cell type-specific inference of differential expression (C-SIDE), that identifies cell type-specific DE in spatial transcriptomics, accounting for localization of other cell......Read more
Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution
Publication: Proceedings of the National Academy of Sciences of the United States of America
Date: Mon, 29 Aug 2022
Authors: Andrew H Laszlo, Jonathan M Craig, Momčilo Gavrilov, Ramreddy Tippana, Ian C Nova, Jesse R Huang, Hwanhee C Kim, Sarah J Abell, Mallory deCampos-Stairiker, Jonathan W Mount, Jasmine L Bowman, Katherine S Baker, Hugh Higinbotham, Dmitriy Bobrovnikov, Taekjip Ha, Jens H Gundlach,
Identifier: pmid:36037333
We used single-molecule picometer-resolution nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of superfamily 1 helicase PcrA as it translocates on, or unwinds, several kilobase-long DNA molecules. We recorded more than two million enzyme steps under various assisting and opposing forces in diverse adenosine tri- and diphosphate conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA......Read more
Structure and engineering of the minimal type VI CRISPR-Cas13bt3
Publication: Molecular cell
Date: Fri, 26 Aug 2022
Authors: Ryoya Nakagawa, Soumya Kannan, Han Altae-Tran, Satoru N Takeda, Atsuhiro Tomita, Hisato Hirano, Tsukasa Kusakizako, Tomohiro Nishizawa, Keitaro Yamashita, Feng Zhang, Hiroshi Nishimasu, Osamu Nureki,
Identifier: pmid:36027912
Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified Cas13bt3 (also known as Cas13X.1) as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the......Read more
How many SARS-CoV-2 "viroporins" are really ion channels?
Publication: Communications biology
Date: Thu, 25 Aug 2022
Authors: Neil L Harrison, Geoffrey W Abbott, Martina Gentzsch, Andrei Aleksandrov, Anna Moroni, Gerhard Thiel, Stephen Grant, Colin G Nichols, Henry A Lester, Andreas Hartel, Kenneth Shepard, David Cabrera Garcia, Masayuki Yazawa,
Identifier: pmid:36008538
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