Lu G, Hickey JW, Haist M
… +23 more, Qin X, Zhao E, Naveed A, Forgo E, Baertsch MA, Mani L, Rovira-Clavé X, Finegersh A, Goltsev Y, Caraccio C, van den Berg NS, Hom M, Colburg DR, Martin BA, Kong CS, Lui NS, Fisher GA, Colevas AD, West RB, Thurber GM, Poultsides GA, Nolan GP, Rosenthal EL
The development of effective antibody therapeutics has been hampered by a lack of methods to measure drug delivery and activity within tumors at single-cell resolution. Here we introduce single-cell spatial pharmacobiolo...The development of effective antibody therapeutics has been hampered by a lack of methods to measure drug delivery and activity within tumors at single-cell resolution. Here we introduce single-cell spatial pharmacobiology (SSP), an experimental and analytical framework that integrates in situ imaging of a systemically infused, fluorescently labeled therapeutic antibody with high-plex spatial proteomics to quantify antibody distribution, target engagement and tumor microenvironment (TME) architecture. We applied SSP to tumor tissues from participants with head and neck squamous cell carcinoma and pancreatic ductal adenocarcinoma who received the antibody panitumumab-IRDye800 in phase 1 trials. SSP identified pronounced spatial heterogeneity in single-cell drug delivery and target engagement, shaped by conserved stromal barriers, including periostin-rich extracellular matrix assemblies and fibroblast-activation-protein-positive cancer-associated fibroblast neighborhoods, which were associated with reduced antibody delivery in both tumor types. SSP measures drug-target-TME interactions in human tumors and can support studies of resistance mechanisms, dosing strategies and discovery of spatial biomarkers for precision oncology.
Cas13-based RNA effectors may enable dynamic, multiplexed and reversible gene regulation in bacteria. Yet, their widespread adoption is hindered by inherent cytotoxicity and collateral cleavage. Here we present a rationa...Cas13-based RNA effectors may enable dynamic, multiplexed and reversible gene regulation in bacteria. Yet, their widespread adoption is hindered by inherent cytotoxicity and collateral cleavage. Here we present a rational protein engineering strategy to generate attenuated Cas13d variants with tunable RNase activity through targeted truncation of flexible regions. This permits effective transcript knockdown while greatly reducing toxicity as reflected by a 2.2-fold higher growth optical density. By introducing proximal mismatches at the 5' end of CRISPR RNA spacers, our system allows functional switching between translation inhibition, polycistronic mRNA degradation and IF3-fusion-based translation-level CRISPR activation. We demonstrate programmable, orthogonal and multiplexed regulation of individual genes within polycistronic mRNAs and synthetic circuits. Application to lycopene biosynthesis optimization shows robust pathway rewiring and improved yields alongside fine-tuned modulation of essential and competing pathways in Escherichia coli. Our work provides a versatile RNA-regulatory toolkit for next-generation microbial synthetic biology and RNA-based biotechnology.
Canarutto D, Fiumara M, Venkatesan V
… +18 more, Gaddoni C, Shiroshita K, Varesi A, Albano L, Pelosi G, Silva A, Firrito C, Schiroli G, Cipria D, Beretta S, Amabile A, Villa A, Zonari E, Gentner B, Lombardo A, Genovese P, Ferrari S, Naldini L
Targeted genomic integration of gene-sized cassettes into hematopoietic stem and progenitor cells (HSPCs) for genetic disease treatment is constrained by the low efficiency of homology-directed repair (HDR) and frequent...Targeted genomic integration of gene-sized cassettes into hematopoietic stem and progenitor cells (HSPCs) for genetic disease treatment is constrained by the low efficiency of homology-directed repair (HDR) and frequent unintended genetic changes at the editing site. Here, to overcome these challenges, we introduce selection by means of artificial transactivators (SMArT), which transiently implements AND reporter gates to achieve templated integration of a functional cassette at the target site. HDR-edited HSPCs were enriched to 80-100% purity through transient selector expression, whereas cells carrying undesired and potentially genotoxic on-target edits were preferentially depleted. Xenotransplantation of SMArT-enriched HSPCs in immunodeficient mice resulted in fully HDR-edited human grafts with the selector no longer detectable. SMArT strategies were implemented through clinically compliant manufacturing and selectors. They support both safe harbor integration and gene correction, can preserve physiological transcriptional regulation and are portable across loci also with polyfunctional editors. Overall, SMArT strategies may broaden the therapeutic applicability of gene-sized editing while reducing its genotoxic burden.
Cellular self-organization reflects an evolutionary leap in which multicellular coordination became essential. Driven by fundamental constraints like oxygen and nutrient transport, physical laws generate inevitable colle...Cellular self-organization reflects an evolutionary leap in which multicellular coordination became essential. Driven by fundamental constraints like oxygen and nutrient transport, physical laws generate inevitable collective behaviors such as cavitation, folding and branching. These behaviors couple mechanics, signaling and gene regulation to build tissues and organs with spatiotemporal precision through the iterative layering of simple rules. Stem cell-based models of embryogenesis and organogenesis make these principles experimentally tractable, revealing canonical developmental routes and alternative trajectories and failure modes that expose bottlenecks and constraints. In this Perspective, we trace self-organization from evolutionary origins to biophysical inevitability and discuss how emerging tools in stem cell biology and bioengineering are beginning to translate these insights into regenerative strategies. Decoding the rules of morphogenesis will open possibilities to reimagine, simulate and rationally engineer the architecture of living tissues.
Optically addressable spin systems have been widely studied for quantum-sensing applications. In this work, we demonstrate that photogenerated spin-correlated radical pairs in certain flavoproteins, cryptochrome and impr...Optically addressable spin systems have been widely studied for quantum-sensing applications. In this work, we demonstrate that photogenerated spin-correlated radical pairs in certain flavoproteins, cryptochrome and improved light-oxygen-voltage protein, can be manipulated by radio waves. This capability enables magnetic field sensing and spatial modulation of photoluminescence using radiofrequency pulses and magnetic field gradients, establishing proteins as a platform for optically addressable spin systems and radiofrequency-based control of biological processes.
Gold MP, Reyes M, Diamant N
… +12 more, Kuo T, Hajiramezanali E, Newburger JW, Son MBF, Lee PY, Scalia G, BenTaieb A, Kapadia SB, Tripathi A, Bravo HC, Heimberg G, Biancalani T
Determining a gene's functional importance within a cellular context has long been a challenge, as absolute expression level is an unreliable indicator. Here we introduce SIGnature, a framework for scoring gene importanc...Determining a gene's functional importance within a cellular context has long been a challenge, as absolute expression level is an unreliable indicator. Here we introduce SIGnature, a framework for scoring gene importance using attributions derived from single-cell RNA-sequencing (scRNA-seq) foundation models. Attribution scores reduce technical noise, emphasize regulatory genes and facilitate cross-dataset comparison-a core challenge for scRNA-seq analyses. We developed the SIGnature package as a tool for generating and querying attributions, enabling rapid gene set searches across large scRNA-seq atlases. We demonstrate its utility using the MS1 monocyte signature, a poorly understood gene program activated in severe COVID-19 and sepsis. Searching 400 studies identified associations between the MS1 signature and multiple hyperinflammatory conditions, including Kawasaki disease. Experimental validation confirmed that serum from persons with Kawasaki disease induces the MS1 phenotype. These findings highlight that SIGnature can uncover shared mechanisms across conditions, demonstrating its power for large-scale signature scoring and cross-disease analysis.
In mass-spectrometry-based proteomics it remains challenging to ensure the accuracy of protein quantities. Here we introduce QuantUMS (quantification using an uncertainty-minimizing solution), a machine learning-based me...In mass-spectrometry-based proteomics it remains challenging to ensure the accuracy of protein quantities. Here we introduce QuantUMS (quantification using an uncertainty-minimizing solution), a machine learning-based method that dynamically tunes the quantification algorithm to minimize quantitative errors. When applied to data-independent acquisition proteomics, QuantUMS increases accuracy and precision, ameliorates ratio compression bias and enhances differential expression analysis. It further reports an uncertainty measure enabling quality control of individual quantities.
Park G, Bian Y, Huang H
… +31 more, Zhou S, Qin S, Lin M, Yang X, Lee A, Ramkumar A, Tome M, Lander J, Chen X, Wang S, Bheemreddy P, Stanton L, Sheng R, Guo G, Shehada M, Wang R, Roa A, Lu C, Yue W, Wu RS, Gao X, Hu H, Yaghi A, Liu M, Impey L, Collins SL, Papageorghiou AT, Laurent LC, Wear KA, Georgieva A, Xu S
Ultrasonography is widely used for fetal monitoring but it requires sonographers and is limited to snapshot evaluations at discrete intervals. Here we report a wearable ultrasound patch (UPatch) for continuous and autono...Ultrasonography is widely used for fetal monitoring but it requires sonographers and is limited to snapshot evaluations at discrete intervals. Here we report a wearable ultrasound patch (UPatch) for continuous and autonomous fetal monitoring. The UPatch can acquire anatomical structures and blood flow velocities, demonstrating good agreement with a handheld clinical ultrasound device on 62 pregnancies. Real-time image segmentation allows autonomous tracking of target vessels to acquire continuous blood flow spectra during fetal and maternal movements without a sonographer. Continuous monitoring data from 52 pregnant women aligned with stratified perinatal conditions, including healthy, small for gestational age, large for gestational age, gestational diabetes, preeclampsia and gestational hypertension. With further technology development, integration with a miniaturized circuit could enable fully wireless operation and greater user mobility. The UPatch could provide continuous assessment of fetal compromise in high-risk pregnancies, expanding prenatal-care capabilities.
Dai Z, Aoki W, Chaiyen P
… +44 more, Chang MW, Chen X, Chen Z, Cho BK, Deng NN, Du S, Foo JL, Fredens J, Fu M, Han X, Hasunuma T, Ichihashi N, Isa NM, Kiga D, Kim DM, Kim J, Kondo A, Kosaka Y, Laoteng K, Lee J, Li J, Li J, Li M, Lim S, Liu J, Liu X, Lu Y, Luo N, Matsuura T, Mizunashi W, Qiao Y, Ramzi AB, Seo SW, Shimizu Y, Shin K, Tian L, Vongsangnak W, Wang B, Zhang CC, Zhang XE, Zhao G, Zheng B, Zhou Y, Liu C
Tao YA, Sakai HA, Jiang AY
… +14 more, Krasnow NA, Vaganov VS, Shim B, Barsdale Z, Pandey S, Ahmed N, Na M, Liao TW, Oye K, Cristian A, Zhang E, Xu JA, Bulcaen M, Liu DR
Although protein engineering and laboratory evolution have been used to optimize prime editors, we show that previous changes that improve prime editor efficiency also compromise protein stability and expression level, l...Although protein engineering and laboratory evolution have been used to optimize prime editors, we show that previous changes that improve prime editor efficiency also compromise protein stability and expression level, limiting performance. To address these limitations, we apply structure-informed artificial intelligence-guided methods such as the inverse-folding network ProteinMPNN to redesign the reverse transcriptase (RT) domains of engineered and evolved prime editors while preserving regions essential for catalysis. Redesigned RTs are extensively mutated, with 30-163 amino acid substitutions, and exhibit enhanced folding stability and soluble expression and up to twofold higher intracellular prime editor protein levels following mRNA delivery. Redesigned PE8 prime editors demonstrate enhanced editing efficiencies across multiple ex vivo contexts, including in several human primary cell types and via several delivery modalities. In mice, editing efficiency is up to 2.9-fold higher than that of state-of-the-art PE6, PE7 and PEmax prime editors. These findings demonstrate a generalizable approach for augmenting laboratory evolution to improve genome editing agents.
The performance of prime-editing (PE) systems has been improved by systematic engineering of their protein and small RNA components but the structured RNA motifs appended to the 3' end of PE guide RNAs (pegRNAs)-a key de...The performance of prime-editing (PE) systems has been improved by systematic engineering of their protein and small RNA components but the structured RNA motifs appended to the 3' end of PE guide RNAs (pegRNAs)-a key determinant of pegRNA stability and editing efficiency-have not been extensively studied. We introduce PE-PRISM, a high-throughput pooled screen to identify and optimize these 3' RNA motifs in human cells. Here, using PE-PRISM, we evaluated 2,858 RNA motifs across four iterative libraries, including natural and engineered pseudoknots, G-quadruplexes and reverse transcriptase recruitment elements. We applied structure-guided mutagenesis and combinatorial variant screening to refine hits, culminating in the engineered and evolved pseudoknot variants tevo2.0, eHAV and eSBRMV1-A. In a screen correcting 847 pathogenic ClinVar variants, the top-performing motifs improved PE efficiency over the widely used tevopreQ motif for >90% of edits. They also increased PE efficiencies for correcting disease-associated mutations in primary human cells and in vivo in mouse brain and liver.
De novo peptide sequencing directly infers sequences from mass spectrometry data without relying on protein databases. Although recent deep learning models can also identify posttranslational modifications (PTMs), they r...De novo peptide sequencing directly infers sequences from mass spectrometry data without relying on protein databases. Although recent deep learning models can also identify posttranslational modifications (PTMs), they require labeled training data for this task. Here we introduce rotary positional embedding-enhanced de novo sequencing algorithm (RNovA), a transformer-based de novo sequencing algorithm enhanced with relative positional embeddings and a reinforcement-learning-style sequential decision framework. RNovA enables open PTM discovery in a zero-shot setting-without retraining or a predefined list of candidate residues-while maintaining state-of-the-art performance on standard benchmarks. Demonstrating this capability, we successfully identified peptides modified by kynurenine-an uncommon and biologically relevant PTM-in clinical samples from patients with RA and validated this discovery with synthetically synthesized reference peptides. Furthermore, we demonstrated open de novo PTM discovery by analyzing the bacterial strain A1232E, which lacks a reference proteome, and detected an unannotated glutamic acid modification. RNovA enables exploration of previously inaccessible regions of the proteome, including peptides with unexpected or unannotated modifications.