CRISPR-Cas effectors typically rely on RNA guides to recognize target sequences. In Cas12a, the protospacer adjacent motif on DNA engages conserved protein residues, triggering target binding and nuclease activation. Her...CRISPR-Cas effectors typically rely on RNA guides to recognize target sequences. In Cas12a, the protospacer adjacent motif on DNA engages conserved protein residues, triggering target binding and nuclease activation. Here we reprogram Cas12a into a DNA-guided, RNA-targeting effector. Exploiting protospacer-adjacent motif-dependent interaction, we engineer synthetic CRISPR DNA that engages Cas12a to form a functional deoxyribonucleoprotein complex, while repurposing solely RNA as the programmable target. Structural, biophysical and biochemical analyses reveal the molecular basis of this DNA-guided, RNA-targeting configuration and support an activation pathway distinct from that of canonical RNA-guided systems. DNA-guided Cas12a enables direct RNA detection and efficient intracellular RNA knockdown, establishing a modular activation architecture for CRISPR-Cas12a and expanding the design space for programmable RNA manipulation.
Wenkel F, Tu W, Masschelein C
… +12 more, Shirzad H, Hodgson L, Bendidi I, Eastwood C, Whitfield ST, Russell C, El Mesbahi Y, Ding J, Fay MM, Earnshaw B, Noutahi E, Denton AK
Accurately predicting cellular responses to genetic perturbations is essential for understanding disease mechanisms and designing effective therapies. Yet, exhaustively exploring the space of possible perturbations (for...Accurately predicting cellular responses to genetic perturbations is essential for understanding disease mechanisms and designing effective therapies. Yet, exhaustively exploring the space of possible perturbations (for example, multigene perturbations or across tissues and cell types) is prohibitively expensive, motivating methods that can generalize to unseen conditions. We present TxPert, a latent-transfer-based deep learning method that uses multiple knowledge graphs of gene (product)-gene (product) relationships to predict transcriptomic perturbation effects. Different knowledge graphs encode complementary information and we show that a combination of graphs derived from biological databases and high-throughput perturbation screens yields the best performance. For predictions of single unseen perturbations, TxPert approaches the performance of split-half experimental reproducibility. For double unseen perturbations and single perturbations in a different cell line, its predictions increase Person Δ for unseen single perturbations by 8-25% over existing methods.
Marks A, Siu S, Bianchini F
… +13 more, Wang C, Lakshmi A, Phelan M, Zhu A, Moon C, Morla-Folch J, Teunissen AJP, Amabile A, Baccarini A, Merad M, Brody JD, Dong Y, Brown BD
Proteins encoded by mRNA vaccines can be expressed by a diversity of transfected cell types but how cell-type-specific expression influences immunity is poorly understood. To investigate this, we incorporated synthetic m...Proteins encoded by mRNA vaccines can be expressed by a diversity of transfected cell types but how cell-type-specific expression influences immunity is poorly understood. To investigate this, we incorporated synthetic microRNA target sites (miRT) into lipid nanoparticle (LNP)-delivered mRNA vaccines to silence mRNA expression specifically in professional antigen-presenting cells (pAPCs), hepatocytes or myocytes. We found that mRNA expression in pAPCs was dispensable for priming antigen-specific T cells, whereas mRNA expression in myocytes induced similar or stronger immune responses, including for SARS-CoV-2, suggesting that antigen cross-presentation or cross-dressing may be more impactful than direct mRNA expression in pAPCs. In contrast, mRNA expression in hepatocytes suppressed the antigen-specific T cell response, partly through PD1/PDL1. In mice bearing tumor-associated antigen (TAA)-expressing lymphoma cells, miRT-mediated hepatocyte-silenced TAA mRNA vaccine enhanced immune response and reduced tumor burden. Thus, non-pAPC expression shapes immunity to mRNA-encoded protein and inclusion of miRTs can boost or blunt mRNA-LNP immunogenicity.
Adeno-associated viruses (AAVs) are preferred gene therapy vectors because of their versatility, durability and safety profile. Here, we demonstrate extensive chimerism, manifesting as pervasive barcode swapping, among c...Adeno-associated viruses (AAVs) are preferred gene therapy vectors because of their versatility, durability and safety profile. Here, we demonstrate extensive chimerism, manifesting as pervasive barcode swapping, among complex recombinant AAV (rAAV) libraries that are packaged as a pool. The observed chimerism is length and homology dependent but capsid independent, in some cases affecting the majority of packaged rAAV genomes. These results have implications for the design and deployment of functional rAAV libraries.
Lipid nanoparticle (LNP) delivery of RNA therapeutics is constrained by poor tissue selectivity and off-target toxicity. Most high-throughput screening approaches have focused on single-target efficacy while overlooking...Lipid nanoparticle (LNP) delivery of RNA therapeutics is constrained by poor tissue selectivity and off-target toxicity. Most high-throughput screening approaches have focused on single-target efficacy while overlooking off-target uptake. Here we report multiobjective LNP engineering with artificial intelligence (MOLEA), a system that integrates high-dimensional lipid representations, cell-type-resolved transfection data and multitask optimization to design ionizable lipids with both high potency and biological selectivity. MOLEA learns structure-function relationships across diverse cellular contexts to identify lipids that preferentially deliver mRNA to target tissue while minimizing hepatocyte transfection. Applying MOLEA to cartilage, we developed K9 LNPs, which achieve >90% transfection efficiency in mouse joint chondrocytes and a 13.5-fold increase in knee-to-liver selectivity compared to the clinical benchmark SM-102. We demonstrate chondrocyte-specific Mmp13 editing in osteoarthritis mouse models, leading to sustained cartilage protection and suppression of disease-associated immune and matrix remodeling. Our findings demonstrate how artificial-intelligence-guided multiobjective optimization can enable precision RNA delivery with potential applications to other tissues.
A common procedure for studying the microbiome is binning the sequenced contigs into metagenome-assembled genomes. State-of-the-art binning methods use coabundance and sequence-based motifs such as tetranucleotide freque...A common procedure for studying the microbiome is binning the sequenced contigs into metagenome-assembled genomes. State-of-the-art binning methods use coabundance and sequence-based motifs such as tetranucleotide frequencies, whereas taxonomic labels derived from alignment based classification have not been widely used. Here we propose TaxVAMB, a metagenome binning tool based on semisupervised bimodal variational autoencoders, combining tetranucleotide frequencies and contig coabundances with taxonomic information. TaxVAMB outperformed all other binners on CAMI2 human microbiome datasets, returning on average 29% more high-quality assemblies than the next best binner, and performed on par with the best binners on short-read datasets. On a human gut long-read dataset, TaxVAMB recovered 29% more high-quality bins. In a typical single-sample setup, TaxVAMB on average returns 83% more high-quality bins compared to VAMB. Lastly, TaxVAMB binned incomplete genomes better than any other tool, returning on average 300% more high-quality bins of incomplete genomes than the next best binner.
Advanced genome editing technologies have enabled rapid, flexible rewriting of the Escherichia coli genome, but most have not been tested in other bacterial species. Recombitrons-a genome editing tool created by pairing...Advanced genome editing technologies have enabled rapid, flexible rewriting of the Escherichia coli genome, but most have not been tested in other bacterial species. Recombitrons-a genome editing tool created by pairing modified, donor-producing bacterial retrons with single-stranded binding and annealing proteins-have increased the efficiency of recombineering to install flexible, precise edits in the prokaryotic chromosome. Here, to extend their utility outside of E. coli, we surveyed the portability and versatility of retron-mediated recombineering across three different bacterial phyla (Proteobacteria, Bacillota and Actinomycetota) and a total of 15 different species. We found that retron recombineering is variable across the species tested but functional in all of them, reaching editing efficiencies >20% in six of them, >40% in three and >90% in two. Efficiencies in the remaining nine species ranged from 0.015% to 7.4%. We also tested the extension of the recombitron architecture, operon and strain modifications in a subset of hosts in which species-specific modifications were required to increase editing rates.
Intravital fluorescence microscopy is hampered by optical aberrations arising from heterogeneous distributions of the refractive index. Adaptive optics (AO) methods are either costly and slow, requiring additional hardwa...Intravital fluorescence microscopy is hampered by optical aberrations arising from heterogeneous distributions of the refractive index. Adaptive optics (AO) methods are either costly and slow, requiring additional hardware, or inaccurate due to lack of wavefront information in multiple angular directions. Here we present a latent-space-enhanced digital AO (LEAO) method that uses wave-optics priors embedded in high-dimensional spatial-angular data and semantically disentangles their representations in the latent space. LEAO achieves more than sixfold higher aberration estimation accuracy than the existing approach (coordinate-based neural representations for computational AO). It also exhibits strong robustness across different system configurations and imaging conditions, achieving almost an order of magnitude higher accuracy than iterative digital AO under extreme conditions such as a low signal-to-noise ratio of 3.4 dB. We experimentally demonstrate that LEAO improves diverse biological observations in vivo, such as large-scale tracking of T cells across an entire lymph node, multiregional neural recording in mouse cortex and long-term monitoring of neutrophil activation, extravasation and clearing through mouse intact skull after traumatic brain injury.
Single-cell RNA sequencing (scRNA-seq) analyses typically focus on alignment and differential gene expression, being blind to other transcriptome variation. Here we present sc-SPLASH for statistics-first, reference-free...Single-cell RNA sequencing (scRNA-seq) analyses typically focus on alignment and differential gene expression, being blind to other transcriptome variation. Here we present sc-SPLASH for statistics-first, reference-free discovery on barcoded scRNA-seq and spatial transcriptomics; its independent BKC submodule optimizes barcoded data preprocessing and is approximately 50-fold faster than UMI-tools. sc-SPLASH discovers secreted repeat proteins in immune-like cells of sponge (Spongilla; missing from the reference) and tunicate (Ciona).
Protein kinases are central to cell signaling and key drug targets in cancer. To inform potential repurposing of kinase inhibitors, we profiled 86 of the ~100 approved kinase inhibitors against 758 kinases, including 409...Protein kinases are central to cell signaling and key drug targets in cancer. To inform potential repurposing of kinase inhibitors, we profiled 86 of the ~100 approved kinase inhibitors against 758 kinases, including 409 wild-type and 349 oncogenic variants using a biochemical kinase assay. Our results increase the number of druggable kinases from 89 to 235, revealing that 94% of mutations and 97% of fusions represented in our samples are inhibited by at least one existing drug. The dataset revealed mutation-specific selectivity, especially in tyrosine kinases FGFR and MET, highlighting gaps and repurposing opportunities. We experimentally validated several actionable findings, including tepotinib to target the IRAK1/4-cholesterol pathway in glioblastoma, brigatinib to target the MARK2/3-Hippo pathway in pancreatic cancer and gilteritinib to overcome MET mutation-driven drug resistance and metastasis. To facilitate exploration of our data, we provide KIRHub, a web-based tool that allows identification of existing inhibitors of wild-type and mutated kinases to guide precision oncology.