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In situ DNA sequencing-based microscopy for subcellular spatial transcriptomics

Project description

A revolutionary sequencing-based microscopy technology

During the early stages of zygote formation, the reservoir of maternal RNA is replaced by the transcription of zygotic genes. To understand the complexity of transcriptional regulation in the embryo, it is necessary to develop new tools for spatial transcriptomics. The EU-funded DNA_MICROSCOPY project will build on a pre-existing 2D topological DNA microscopy approach that performs in situ PCR and next-generation sequencing. Researchers aim to perform in situ whole-transcriptome sequencing in 3D with subcellular resolution and combine it with protein analysis. The novel technology will help dissect key events of early development, such as the determination of cell polarity and fate during cleavage.

Objective

New tools are needed in spatial transcriptomics, which uses imaging to resolve the positions of RNA in their native biological contexts to reveal molecular mechanisms underlying cell states and interactions. The developing embryo exhibits complex transcriptional regulation during the maternal-to-zygotic transition, when the reservoir of maternal RNA is phased out and the zygotic genes are turned on. Existing spatial sequencing approaches cannot simultaneously achieve subcellular resolution, whole transcriptome coverage, isotropic 3D resolution, and the parallel mapping of proteins and genetic regulatory elements that is desirable to form a mechanistic picture of transcriptional regulation in any system as complex is the embryo. The nascent field of DNA microscopy is perfectly suited for the high-throughput, multiplexed, molecular mapping needs of such problems. DNA microscopy uses carefully engineered in situ PCR, next-generation sequencing, and the mathematics of stochastic geometry instead of optics to convey microscale spatial information. In 2018, I developed a 2D DNA microscopy approach based on topological reconstruction of adjacent patches of barcoded DNA polonies. My work is among a few papers appearing just in the last year that together constitute a new field. I will adapt my 2D topological DNA microscopy method to one based on in situ whole-transcriptome sequencing in 3D, aiming to achieve subcellular resolution. I will also develop the mathematical basis of topological reconstruction and new computational tools to deal with the 3D data. Finally, I aim to incorporate the capability to localize other molecules in parallel with the transcriptome such as oligonucleotide-conjugated antibodies targeting specific transcription factors and other genetic regulatory elements. The technique, deployed on developing C. elegans embryos, will be used to study spatially dependent regulatory mechanisms such as the determination of cell polarity and fate during cleavage in greater breadth and depth than ever previously achieved. By being optics-free, this has the potential to overcome fundamental limitations imposed by traditional forms of microscopy, greatly expand the ease and throughput of spatial transcriptomics, and pave the way for routine use of hyper-multiplexed molecular imaging.

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Topic(s)

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ERC-STG - Starting Grant

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Call for proposal

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(opens in new window) ERC-2020-STG

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Host institution

KUNGLIGA TEKNISKA HOEGSKOLAN
Net EU contribution

Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.

€ 1 499 947,00
Address
BRINELLVAGEN 8
100 44 STOCKHOLM
Sweden

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Region
Östra Sverige Stockholm Stockholms län
Activity type
Higher or Secondary Education Establishments
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Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

€ 1 499 947,00

Beneficiaries (1)

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