Method of in situ gene sequencing
Inventors
Wang, Xiao • Allen, William E. • Deisseroth, Karl A.
Assignees
Leland Stanford Junior University
Publication Number
US-12359253-B2
Publication Date
2025-07-15
Expiration Date
2039-04-04
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Abstract
Provided herein are devices, methods, and systems for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue. Methods of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue are also provided herein.
Core Innovation
The invention provides devices, methods, and systems for performing in situ gene sequencing of a target nucleic acid within cells in intact tissues, enabling spatially-resolved transcriptomics with subcellular resolution. The disclosed methods involve contacting fixed and permeabilized intact tissues with pairs of oligonucleotide primers that specifically hybridize to target nucleic acids, followed by ligation to generate closed nucleic acid circles, rolling circle amplification to form amplicons, embedding the amplicons in hydrogels, performing multiple cycles of sequencing-by-ligation using specialized primers, and imaging to determine gene sequences in situ.
The problem addressed by this patent arises from the challenges inherent in implementing enzymatic reactions for sequencing in dense, complex tissue environments, where existing sequencing methods suffer from low efficiency, insufficient sensitivity, fidelity, and scalability, particularly in three-dimensional volumes of intact tissue such as mammalian brain. Current approaches fail to retrieve high-content gene-expression information while retaining three-dimensional positional anatomy at cellular resolution, limiting integrative understanding of tissue structure and function, especially in complex tissues with diverse cell types.
The invention introduces a method integrating specific signal amplification via Specific Amplification of Nucleic Acids via Intramolecular Ligation (SNAIL), hydrogel-tissue chemistry for embedding and clearing, and a novel sequencing-by-ligation strategy called Sequencing with Error-Correction by Dynamic Annealing and Ligation (SEDAL). The SNAIL method improves specificity and efficiency by utilizing pairs of oligonucleotide primers that hybridize adjacently to target nucleic acids, facilitating intramolecular ligation and generation of amplicons with barcode sequences for identification. Hydrogel embedding stabilizes amplicons and allows clearing of lipids and proteins to enhance optical clarity and reagent diffusion. SEDAL provides highly accurate sequencing at room temperature with error reduction.
Claims Coverage
The claims encompass two independent methods and a system, covering in situ identification and sequencing of nucleic acids in tissue and screening of candidate agents for modulation of gene expression.
Specific hybridization of oligonucleotide pairs to target nucleic acids in tissue
A pair of oligonucleotides, each comprising three complementarity regions, hybridizes adjacently to a target nucleic acid in fixed, permeabilized tissue to allow for specific binding and formation of linkage.
Ligation and rolling circle amplification to generate amplicons
Use of ligase to circularize the second oligonucleotide and rolling circle amplification using the second oligonucleotide as template and the first as primer to produce hydrogel-embedded amplicons representing the target nucleic acid.
Hydrogel embedding and tissue clearing
Embedding amplicons in a hydrogel via copolymerization (e.g., with acrylamide), allowing clearing of cellular components (such as lipids and proteins) to retain nucleic acid signals while providing optical transparency and structural preservation.
Sequencing-by-ligation with error correction
Performing sequencing cycles by contacting hydrogel-embedded amplicons with pairs of primers (third and fourth oligonucleotide) that ligate only when perfectly matched, enabling dynamic annealing and ligation (SEDAL) to provide high-fidelity sequence determination with multiple reiterated ligation steps and fluorescent imaging.
Imaging using advanced microscopy techniques
Imaging embedded amplicons using microscopy such as confocal, two-photon, light-field, intact tissue expansion microscopy, or CLARITY-optimized light sheet microscopy to determine in situ gene sequence spatially.
Screening method for candidate agents modulating gene expression
Performing the in situ sequencing methods above to detect gene expression levels in presence and absence of candidate agents in intact tissue, where alterations in expression indicate modulation by the agent.
System for automated sequencing
A system comprising a fluidics device with imaging chambers and pump, and a processor configured to perform in situ gene sequencing methods, thus enabling automation of sequencing chemistry delivery, image acquisition, and reagent cycling.
The claims collectively cover a comprehensive methodology integrating specific hybridization, ligation, rolling circle amplification, hydrogel embedding with clearing, error-correcting sequencing-by-ligation, and imaging for in situ gene sequencing in intact tissues, as well as applying these methods for screening gene expression modulation and automation in an integrated system.
Stated Advantages
Faster processing time compared to existing microarray or sequencing technologies, allowing completion in approximately three to four days from raw sample to data.
High multiplexing capability, allowing detection and sequencing of up to 1000 genes simultaneously.
High efficiency and sensitivity, enabling single-cell and single-molecule resolution with enhanced signal-to-noise ratio and low error rates.
Preservation of tissue morphology and three-dimensional architecture during sequencing, allowing spatial mapping in intact tissues.
Error reduction in sequencing by the novel sequencing-by-ligation methodology (SEDAL) implemented at room temperature for low background noise.
Automation potential through fluidics systems that coordinate reagent delivery and imaging, enabling continual operation and reproducibility.
Documented Applications
Spatially resolved gene expression analysis in biological research for fundamental biology and drug screening.
Clinical diagnostics including detection of gene markers related to diseases, immune responses, bacterial or viral infections in patient samples.
Identification and classification of diverse molecularly-defined cell types in mouse brain, including cortical neurons and glial subtypes, with spatial organization insights.
Measurement of activity-regulated gene expression changes in response to sensory stimuli in neural tissues.
Quantitative three-dimensional analysis of large tissue volumes at cellular resolution.
Screening of candidate therapeutic agents for their modulatory effects on gene expression within intact tissues.
Potential use in personalized medicine through detection of genetic markers, chromosomal abnormalities, and therapy responsiveness.
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