How to build the virtual cell with artificial intelligence: Priorities and opportunities

Cell, Journal Year: 2024, Volume and Issue: 187(25), P. 7045 - 7063

Published: Dec. 1, 2024

Cells are essential to understanding health and disease, yet traditional models fall short of modeling simulating their function behavior. Advances in AI omics offer groundbreaking opportunities create an virtual cell (AIVC), a multi-scale, multi-modal large-neural-network-based model that can represent simulate the behavior molecules, cells, tissues across diverse states. This Perspective provides vision on design how collaborative efforts build AIVCs will transform biological research by allowing high-fidelity simulations, accelerating discoveries, guiding experimental studies, offering new for cellular functions fostering interdisciplinary collaborations open science.

Language: Английский

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CROPseq-multi: a versatile solution for multiplexed perturbation and decoding in pooled CRISPR screens

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 17, 2024

Forward genetic screens seek to dissect complex biological systems by systematically perturbing elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves performance of single-target enables combinatorial for study interactions. Current tools are incompatible with pooled that require mRNA-embedded barcodes, including some microscopy single cell sequencing approaches. Here, we report development CROPseq-multi, a CROPseq-inspired lentiviral system multiplex Streptococcus pyogenes (Sp) Cas9-based barcodes. CROPseq-multi has equivalent per-guide activity CROPseq low recombination frequencies. is compatible enrichment optical screens, extensible single-cell readouts. For an optimized multiplexed in situ detection protocol barcode efficiency 10-fold, events, increases decoding 3-fold relative CROPseq. widely applicable solution diverse SpCas9-based

Language: Английский

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Massively parallel in vivo Perturb-seq screening

Nature Protocols, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 12, 2025

Language: Английский

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Simultaneous CRISPR screening and spatial transcriptomics reveal intracellular, intercellular, and functional transcriptional circuits

Cell, Journal Year: 2025, Volume and Issue: unknown

Published: March 1, 2025

Highlights•Perturb-FISH combines spatial transcriptomics and readout of CRISPR perturbation•We recover the effects genetic perturbation on transcriptome single cells•We find specific networks related to cell neighbors in tissue•We connect time-resolved imaging phenotypes after perturbationSummaryPooled optical screens have enabled study cellular interactions, morphology, or dynamics at massive scale, but they not yet leveraged power highly plexed single-cell resolved transcriptomic readouts inform molecular pathways. Here, we present a combination with parallel detection situ amplified guide RNAs (Perturb-FISH). Perturb-FISH recovers intracellular that are consistent RNA-sequencing-based (Perturb-seq) screen lipopolysaccharide response cultured monocytes, it uncovers intercellular density-dependent regulation innate immune response. Similarly, three-dimensional xenograft models, identifies tumor-immune interactions altered by knockout. When paired functional separate autism spectrum disorder risk genes human-induced pluripotent stem (hIPSC) astrocytes, shows common calcium activity their associated dysregulated is thus general method for studying associations biology resolution.Graphical abstract

Language: Английский

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Multimodal scanning of genetic variants with base and prime editing

Nature Biotechnology, Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 12, 2024

Mutational scanning connects genetic variants to phenotype, enabling the interrogation of protein functions, interactions and variant pathogenicity. However, current methodologies cannot efficiently engineer customizable sets diverse in endogenous loci across cellular contexts high throughput. Here, we combine cytosine adenine base editors a prime editor assess pathogenicity broad spectrum epithelial growth factor receptor gene (EGFR). Using pooled editing guide RNA libraries, install tens thousands spanning full coding sequence EGFR multiple cell lines role these tumorigenesis resistance tyrosine kinase inhibitors. Our scan identifies important hits, supporting robustness approach revealing underappreciated routes activation drug response. We anticipate that multimodal precision mutational can be applied broadly characterize variation any element interest at single-nucleotide resolution.

Language: Английский

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Optical Pooled Screening for the Discovery of Regulators of the Alternative Lengthening of Telomeres Pathway

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 16, 2025

Telomere elongation is essential for the proliferation of cancer cells. length control achieved by either activation telomerase enzyme or recombination-based Alternative Lengthening Telomeres (ALT) pathway. ALT active in about 10-15% human cancers, but its molecular underpinnings remain poorly understood, preventing discovery potential novel therapeutic targets. Pooled CRISPR-based functional genomic screens enable unbiased factors involved biology. Recently, Optical Screens (OPS) have significantly extended capabilities pooled genomics to sensitive imaging-based readouts at single cell level and large scale. To gain a better understanding pathway, we developed OPS assay that employs telomeric native DNA FISH (nFISH) as an optical quantitative readout measure activity. The uses standard protocols library preparation sequencing. As critical element, optimized nFISH protocol performed before situ sequencing maximize performance. We show modified faithfully detects changes activity upon CRISPR knock-out (KO) FANCM BLM genes which were previously implicated ALT. Overall, OPS-nFISH reliable method can provide deep insights into pathway high-throughput format.

Language: Английский

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CellFIE: Integrating Pathway Discovery With Pooled Profiling of Perturbations Uncovers Pathways of Huntington's Disease, Including Genetic Modifiers of Neuronal Development and Morphology

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 20, 2025

Abstract Genomic screens and GWAS are powerful tools for identifying disease-modifying genes, but it is often challenging to understand the pathways by which these genes function. Here, we take an integrated approach that combines network analysis imaging-based pooled genetic perturbation study examine modifiers of Huntington’s disease (HD). The computational highlighted several in a subnetwork enriched neuronal development morphology. To test functional roles developed experimental pipeline allows CRISPRi KD 21 human iPSC-derived neurons followed optical genotypes, arborization, multiplexed pathway activity morphological fingerprint readout. This recovered known involved morphology confirmed unexpected links from between HD Our overcomes challenges measurement function health could be adapted other phenotypes neurological diseases.

Language: Английский

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PerturbView: scalable image-based perturbation screens in cells and tissues

Nature reviews. Immunology, Journal Year: 2025, Volume and Issue: unknown

Published: March 5, 2025

Language: Английский

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Sequencing-free whole genome spatial transcriptomics at molecular resolution in intact tissue

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: March 11, 2025

Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, compositions, interactions, organizations, and functions. Yet existing tools are still limited either transcriptomic coverage or resolution. Leading spatial-capture spatial-tagging techniques that rely on in-vitro sequencing offer whole-transcriptome coverage, principle, but at the cost lower resolution compared to image-based techniques. In contrast, high-performance techniques, which situ hybridization sequencing, achieve single-molecule retain sub-cellular morphologies, by probe libraries target only a subset transcriptome, typically covering several hundred few thousand transcript species. Together, these limitations hinder unbiased, hypothesis-free analyses high Here we develop new technology termed Reverse-padlock Amplicon Encoding FISH (RAEFISH) with whole-genome level while retaining intact tissues. We demonstrate targeting 23,000 human species 22,000 mouse species, including nearly entire protein-coding transcriptome long-noncoding RNAs, single cells cultures tissue sections. Our reveal differential subcellular localizations transcripts, cell-type-specific cell-type-invariant zonation dependent gene expression programs underlying preferential cell-cell interactions. Finally, further for direct readout gRNAs an high-content CRISPR screen. Overall, developments provide research community broadly applicable enables high-coverage, high-resolution profiling both long short, native engineered RNA many biomedical contexts.

Language: Английский

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Transcriptome-wide analysis of differential expression in perturbation atlases

Nature Genetics, Journal Year: 2025, Volume and Issue: unknown

Published: April 21, 2025

Language: Английский

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