Several high-throughput sequencing methods have been used to study the genome-wide chromatin occupancy of long noncoding RNAs (lncRNAs), including ChIRP-seq, CHART-seq and RAP-seq. Many of the datasets obtained with thes...Several high-throughput sequencing methods have been used to study the genome-wide chromatin occupancy of long noncoding RNAs (lncRNAs), including ChIRP-seq, CHART-seq and RAP-seq. Many of the datasets obtained with these methods contain thousands of binding sites, which appears to be in contradiction with the low abundance of the interrogated lncRNAs. Here, we study the chromatin interactome of NESPR lncRNA in cells with varying levels of endogenous expression and perform a meta-analysis using dozens of RNA-chromatin interaction datasets in human and mouse cells. We demonstrate that thousands of regions reported to bind lncRNAs most likely arise from the spurious recovery of DNA elements, where the ends of the recovered DNA fragments exhibit partial complementarity with the probes used for the pulldown. In addition, crucial controls were rarely used in previous studies. Therefore, most chromatin regions reported as bound by trans-acting RNAs in recent studies in mammalian cells appear to be technical artifacts. We provide suggestions for assessing the quality of RNA-chromatin datasets and their improvement.
Orosco C, Huang B, Rananaware SR
… +14 more, Bodin AP, Browning I, Fang A, Baugh MP, Lange IH, Elhabashy YB, Balaraju M, Lewis JG, Shah NH, Hanna MP, Flannery SJ, Meister KS, Karalkar V, Jain PK
Here, we present ΨDNA, a DNA-based guide that enables RNA targeting by Cas12 nucleases, overcoming the traditional reliance on RNA-guided systems. We engineer ΨDNA to mimic a CRISPR RNA (crRNA) scaffold in reverse orient...Here, we present ΨDNA, a DNA-based guide that enables RNA targeting by Cas12 nucleases, overcoming the traditional reliance on RNA-guided systems. We engineer ΨDNA to mimic a CRISPR RNA (crRNA) scaffold in reverse orientation, allowing AsCas12a and Cas12i1 to recognize RNA and trigger strong single-stranded DNA trans-cleavage for sensitive detection of diverse RNA species, including 100% accurate hepatitis C virus RNA detection in clinical samples. ΨDNA also achieves 70-95% multiplex knockdown of endogenous intracellular RNA transcripts through ribosome stalling across multiple human cell lines. Mechanistic studies reveal that activity depends on a stem loop that stabilizes a catalytically competent Cas12-ΨDNA-RNA complex. Lastly, codelivery of crRNA and ΨDNA enables simultaneous DNA editing and RNA knockdown with a single effector and modular fusions of different enzymes to AsCas12a extend ΨDNA to RNase H-mediated RNA degradation and METTL3-based epitranscriptomic editing. Together, ΨDNA guides constitute an adaptable toolkit that extends Cas12 systems beyond genome editing and diagnostics to enable precise, programmable control of cellular transcriptomes and their epitranscriptomic marks.
Predicting T cell receptor (TCR) specificity on the basis of sequence is challenging because TCRs of similar sequence can recognize entirely different antigens, whereas TCRs of different sequence can recognize the same a...Predicting T cell receptor (TCR) specificity on the basis of sequence is challenging because TCRs of similar sequence can recognize entirely different antigens, whereas TCRs of different sequence can recognize the same antigens. Here we present a system that integrates high-throughput yeast display with fine-tuned protein language models (pLMs) to generate deep peptide recognition profiles (PRPs) for individual TCRs, each detailing binding against millions of peptides. We provide detailed PRPs for a panel of HLA-B*27:05-restricted TCRs from persons with ankylosing spondylitis and acute anterior uveitis that almost exclusively recognize peptides through CDR3β. pLMs trained on these PRPs outperform AlphaFold3 and tFold-TCR in predicting T cell activation. We discover and validate novel candidate autoantigens, demonstrate that model generalization to new TCRs correlates with functional distance (PRP divergence) rather than sequence similarity and introduce a model-intrinsic uncertainty metric to quantify prediction confidence. This system and its associated PRP datasets offer a scalable approach to mapping TCR recognition, accelerating antigen discovery and guiding TCR engineering.
Gupta A, Das R, Reed K
… +10 more, Jeon T, Nguyen QTC, Rudra A, Ge X, Trongjit S, Vanrobaeys YS, Langer R, Weissleder R, Garris C, Anderson DG
Nat Biotechnol
· 2026 May · PMID 42129506
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Although immunotherapy has benefited a subset of persons with cancer, its broader efficacy remains limited, primarily because of an immunosuppressive tumor microenvironment characterized by insufficient numbers of functi...Although immunotherapy has benefited a subset of persons with cancer, its broader efficacy remains limited, primarily because of an immunosuppressive tumor microenvironment characterized by insufficient numbers of functional tumor-specific T cells, antigen-presenting cells (APCs) and tumor-infiltrating lymphocytes. Here we engineer immune cells in the tumor microenvironment using lipid nanoparticles (LNPs) to deliver immune-remodeling mRNAs (IR-mRNAs) encoding NF-κB-inducing kinase or interferon regulatory factor 8. These IR-mRNAs activate APCs in tumors, significantly increasing activated type 1 conventional dendritic cells, immunostimulatory cytokines and priming antitumor CD8 T cells. IR-mRNAs encapsulated in LNPs elicited durable antitumor responses in multiple syngeneic mouse tumor models through both intratumoral and intravenous delivery. Coadministration of IR-mRNA and ovalbumin mRNA elicited a ~10-fold increase in antigen-specific CD8 T cell responses, sustained long-term memory and effectively prevented tumor growth in vaccinated mice. Additionally, coadministration of IR-mRNA and hemagglutinin mRNA enhanced the humoral response ~5-fold and the cellular response ~15-fold, underscoring their potential as adjuvants for boosting adaptive immunity.
Elucidating gene function in highly redundant genetic programs such as signaling pathways is challenging in model and nonmodel plants with current whole-plant genetic screening tools. Many of these challenges could be ov...Elucidating gene function in highly redundant genetic programs such as signaling pathways is challenging in model and nonmodel plants with current whole-plant genetic screening tools. Many of these challenges could be overcome if screens were instead carried out using individual cells harboring genetic perturbations. Here we report a single-cell screening platform, PIVOT (protoplast isolation after virus overexpression in planta), to accelerate identification and functional characterization of plant genes. We use Nicotiana benthamiana as a heterologous host to test gene libraries arrayed in a single leaf. PIVOT harnesses viral superinfection exclusion to ensure single multiplicity of infection per cell during pooled library delivery. Additionally, we engineer a cell-surface protein as a phenotypic marker for isolating cells of interest from a heterogeneous population. Using this system, we recover regulators of cytokinin signaling from an Arabidopsis open reading frame library. We anticipate PIVOT will be broadly applicable for high-throughput, single-cell functional genetic screening across the plant kingdom.
Zhou X, Wang B, Wei Y
… +16 more, Hacker S, Kim S, Borrillo T, McCaig A, Ahmed H, Ren Y, Hough O, Orsini L, Chao BT, McInnis M, Cypel M, Liu M, Yeung JC, Del Sorbo L, Keshavjee S, Sage AT
Digital twins are an emerging concept in healthcare that envisions integration of molecular, physiological, functional and clinical data to create computational models of biological systems such as cells, organs and indi...Digital twins are an emerging concept in healthcare that envisions integration of molecular, physiological, functional and clinical data to create computational models of biological systems such as cells, organs and individuals. However, the lack of large, multimodal datasets has so far precluded the realization of comprehensive digital twins in medicine. Ex vivo lung perfusion (EVLP) allows the study of human lungs outside the body under physiological conditions and generates multimodal data from imaging, physiologic monitoring and molecular assays. Here we report lung digital twins developed from the largest known clinical EVLP dataset. We show that the digital twin framework accurately models >75 parameters spanning lung physiology, biochemistry, radiography, transcriptomics, metabolomics and proteomics. Furthermore, direct comparison to experimental data on EVLP lungs treated with alteplase demonstrates that digital twins can precisely assess therapeutic efficacy. Together, these results establish human lung digital twins developed using EVLP as a data-rich approach to improve the evaluation of therapeutic effects.