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CAT-seq: A Chemical Strategy for Subcellular-Resolved RNA Profiling and Synchronous Multi-omics Investigation

ABSTRACT: Understanding cellular functions in health and disease requires dissecting spatiotemporal variations in the subcellular transcriptome. Mitochondria, with their independent RNA metabolism, play pivotal roles in numerous biological processes. Existing methods for mitochondrial RNA profiling suffer from limitations such as low resolution, contamination, and dependence on genetic manipulation. Here, we present CAT-seq, a bioorthogonal photocatalytic labeling strategy that enables high-resolution, in situ profiling of mitochondrial RNA in living cells without genetic manipulation. Through systematic exploration of quinone methide warheads, we identified an efficient RNA labeling probe. Rigorous validation and optimization enabled CAT-seq to successfully profile mitochondrial RNA, track RNA dynamics in HeLa cells, and even the challenging RAW 264.7 macrophages, achieving ~60% specificity. Furthermore, leveraging the unique reactivity of distinct quinone methides, we established an orthogonal labeling system enabling synchronous RNA and protein multi-omics profiling within the same sample. This allows us to unravel the intricate link between mitochondrial RNA and protein changes. Applying synchronous multi-omics to RAW 264.7 cells during immune response revealed an underlying mitochondrial remodeling mechanism behind oxidative phosphorylation pathway reduction. By integrating bioorthogonal photocatalytic chemistry with proximity-based labeling, CAT-seq offers a general, catalytic, and non-genetic approach for subcellular RNA and multi-omics investigations. This opens up exciting avenues for studying diverse physiological and pathological processes with RNA involvement.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE255007 | GEO | 2025/06/24

REPOSITORIES: GEO

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Project description:Rare cells exert substantial impact on tissue physiology and pathology across a spectrum of biological realms. The elucidation of their physiological functions through multi-omics analysis is pivotal. Nonetheless, advancements in trace-level cell multi-omics technology are impeded by cellular loss during experimental procedures. Mitochondrial DNA (mtDNA) editing facilitates disease modeling of mitochondrial genetic disorders in cell lines and animals, with potential for future therapeutic applications. However, the absence of technology capable of simultaneously assessing the efficiency of mitochondrial gene editors and molecular phenotypes limits the development of mitochondrial gene editors and their in vivo therapeutic applications. Here, to address these challenges, we devised a novel omics carrier microparticle, abbreviated as OmicsCam for driving low-input cells multi-omics. OmicsCam consists of three key components: miniaturized open microparticles for multi-step biochemistry, an enlarged cell chamber boundary for streamline manipulation and minimize cell loss, and tunable permeability to ensure compatibility with key steps of magnetic separation in automated systems. By refining OmicsCam's manufacturing process, optimizing cell permeation conditions, and fine-tuning multi-omics library biochemistry, we demonstrated simultaneous assessment of mtDNA editing efficiency (mtDNA sequencing), post-editing cellular transcriptome, and chromatin accessibility in minute cell samples containing as few as 25,000 cells. Moreover, we can concurrently analyze off-target effects of gene editing. The OmicsCam platform offers a powerful way for microscale cell multi-omics analysis, enabling interrogation mitochondrial gene editing efficiency and molecular phenotypes in a unified framework. This offers a holistic perspective on the consequences of genetic manipulation within the mitochondrial genome, thereby advancing our understanding of mitochondrial biology and facilitating the development of precision therapeutics for mitochondrial disorders.

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CAT-seq: A Chemical Strategy for Subcellular-Resolved RNA Profiling and Synchronous Multi-omics Investigation

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