Spatial transcriptomics for profiling the tropism of viral vectors in tissues

Jang, Min Jee; Coughlin, Gerard M; Jackson, Cameron; Chen, Xinhong; Chuapoco, Miguel; Vendemiatti, Julia; Wang, Alexander; Gradinaru, Viviana

doi:10.1038/s41587-022-01648-w

Cited by 29 publications

(28 citation statements)

References 93 publications

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“…mRNA exclusively appeared in the cells expressing EGFP (Figure3, upper row). Additionally, HCR FISH for EGFP mRNA was tested in slices of Thy1-EGFP mice treated with the passive clarity technique (PACT),25 and the results exhibited HCR FISH signal within cells where EGFP fluorescent signal was detectable (Figure3, lower row), in accordance with cell test results.…”

supporting

confidence: 65%

“…Therefore, enhancing the imaging signal intensity for tissue imaging became imperative. To achieve this, the in situ rolling circle amplification (ISRCA) method based on SNAIL probes was combined with HCR to further amplify HCR signals. , The SNAIL probes comprise a phosphorylated padlock and primer. The padlock forms a single strand DNA circle when complementarily paired with the primer sequence and ligated by T4 DNA ligase (Figure A, upper panel).…”

Section: Resultsmentioning

confidence: 99%

“…Here, we combined ISRCA with HCR, resulting in a substantial enhancement of the FISH signal. ISRCA offers unparalleled superiority for multiplex detection, capable of simultaneous labeling of over a hundred genes. ,, In this study, we realized signal amplification and multiplex detection through the combination of HCR and ISRCA. In comparison with similar techniques, such as the RNAscope method, which utilizes bDNA amplification, the amplification capacity of RNAscope is restricted.…”

Section: Discussionmentioning

confidence: 99%

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Designing a Hybrid Chain Reaction Probe for Multiplex Transcripts Assay with High-Level Imaging

Cao,

Qin,

Wang

et al. 2023

ACS Nano

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The hybrid chain reaction (HCR), an isothermal and enzyme-free amplification strategy, has found extensive use in fluorescent in situ hybridization (FISH) assays. However, the existing HCRs are limited, being time-consuming processes and low-efficiency imaging due to weak signal, significantly restricting their application in transcriptomic assays. To address the limitations, we developed nine orthogonal HCR hairpin-pair (hp) probes in this study to enable efficient signal amplification for multiplex assays. To enhance the efficiency and imaging quality of multiplex assays using these HCR probes, we employed two strategies. First, we coupled fluorescent molecules to HCR hairpins via disulfide bonds, facilitating easy removal through chemical cleavage. As a result, the workflow was greatly simplified. Second, we combined HCR with in situ rolling circle amplification (ISRCA), creating ISRCA-HCR, which achieved a 17-fold signal amplification. ISRCA-HCR demonstrated a high-level imaging capability for spatial cell type assays. This study shows the application for cell typing based on the developed HCR probes, enabling accurate and high-level signal amplification for multiplex FISH imaging. This provides an effective research tool for transcriptome and spatial cell type analysis.

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supporting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Designing a Hybrid Chain Reaction Probe for Multiplex Transcripts Assay with High-Level Imaging

Cao,

Qin,

Wang

et al. 2023

ACS Nano

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“…Methods to target and manipulate mammalian cell types in vivo have been revolutionized in the past decade. Building on these efforts, a key advantage of this massively parallel in vivo Perturb-seq platform is its modularity: diverse serotypes and capsids of AAV can allow cell type- and tissue-specific targeting of even rare cell populations in vivo , expanding Perturb-seq from rodents to additional animal models (Chuapoco et al, 2023; Jang et al, 2023; Tabebordbar et al, 2021). However, AAV transgene expression could be diluted or eventually lost, depending on the cell division and turnover rate in vivo .…”

Section: Discussionmentioning

confidence: 99%

Massively parallelin vivoPerturb-seq reveals cell type-specific transcriptional networks in cortical development

Zheng,

Wu,

Liu

et al. 2023

Preprint

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Systematic analysis of gene function across diverse cell types in vivo is hindered by two challenges: obtaining sufficient cells from live tissues and accurately identifying each cell's perturbation in high-throughput single-cell assays. Leveraging AAV's versatile cell type tropism and high labeling capacity, we expanded the resolution and scale of in vivo CRISPR screens: allowing phenotypic analysis at single-cell resolution across a multitude of cell types in the embryonic brain, adult brain, and peripheral nervous system. We undertook extensive tests of 86 AAV serotypes, combined with a transposon system, to substantially amplify labeling and accelerate in vivo gene delivery from weeks to days. Using this platform, we performed an in utero genetic screen as proof-of-principle and identified pleiotropic regulatory networks of Foxg1 in cortical development, including Layer 6 corticothalamic neurons where it tightly controls distinct networks essential for cell fate specification. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% (mediated by lentivirus), and achieve analysis of over 30,000 cells in one experiment, thus enabling massively parallel in vivo Perturb-seq. Compatible with various perturbation techniques (CRISPRa/i) and phenotypic measurements (single-cell or spatial multi-omics), our platform presents a flexible, modular approach to interrogate gene function across diverse cell types in vivo, connecting gene variants to their causal functions.

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“…In this evolving landscape, we present Patho-DBiT not only enabling spatial full-coverage base-by-base whole transcriptome sequencing but also meticulously crafted to address the distinctive challenges of clinically archived FFPE tissues (Figure 1A). Patho-DBiT integrates in situ polyadenylation 10,11 , deterministic barcoding in tissue (DBiT) using microfluidic chips 12 , and computational innovations to navigate and decode the rich RNA biology inherent in FFPE samples. The platform adeptly capitalizes on RNA fragmentation naturally occurring in FFPE specimens and appends poly(A) tails to a broad spectrum of RNA species, thereby overcoming traditional barriers associated with FFPE samples and even outperforming the assays conducted with fresh frozen tissues.…”

Section: Introductionmentioning

confidence: 99%

Spatially Exploring RNA Biology in Archival Formalin-Fixed Paraffin-Embedded Tissues

Bai,

Zhang,

Gao

et al. 2024

Preprint

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Spatial transcriptomics has emerged as a powerful tool for dissecting spatial cellular heterogeneity but as of today is largely limited to gene expression analysis. Yet, the life of RNA molecules is multifaceted and dynamic, requiring spatial profiling of different RNA species throughout the life cycle to delve into the intricate RNA biology in complex tissues. Human disease-relevant tissues are commonly preserved as formalin-fixed and paraffin-embedded (FFPE) blocks, representing an important resource for human tissue specimens. The capability to spatially explore RNA biology in FFPE tissues holds transformative potential for human biology research and clinical histopathology. Here, we present Patho-DBiT combiningin situpolyadenylation and deterministic barcoding for spatial full coverage transcriptome sequencing, tailored for probing the diverse landscape of RNA species even in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for five years. Furthermore, genome-wide single nucleotide RNA variants can be captured to distinguish different malignant clones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA-mRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis trajectory. High resolution Patho-DBiT at the cellular level reveals a spatial neighborhood and traces the spatiotemporal kinetics driving tumor progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to study human tissue biology and aid in clinical pathology evaluation.

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Spatial transcriptomics for profiling the tropism of viral vectors in tissues

Cited by 29 publications

References 93 publications

Designing a Hybrid Chain Reaction Probe for Multiplex Transcripts Assay with High-Level Imaging

Designing a Hybrid Chain Reaction Probe for Multiplex Transcripts Assay with High-Level Imaging

Massively parallelin vivoPerturb-seq reveals cell type-specific transcriptional networks in cortical development

Spatially Exploring RNA Biology in Archival Formalin-Fixed Paraffin-Embedded Tissues

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