156: RAEFISH: Sequencing-free whole-genome spatial transcriptomics at single-molecule resolution

️ Episode 156: RAEFISH: Sequencing-free whole-genome spatial transcriptomics at single-molecule resolution In this episode of PaperCast Base by Base, we explore RAEFISH, a reverse-padlock amplicon-encoding FISH method that delivers whole-transcriptome imaging at single-molecule resolution without sequencing. The study demonstrates genome-scale coverage across cells and intact tissues and extends to direct readout of CRISPR guide RNAs, enabling high-content functional screens with spatial context. Study Highlights:RAEFISH introduces a “reversed” padlock design with splint-assisted ligation, rolling-circle amplification, and MERFISH-style sequential readouts to barcode >20,000 transcripts while remaining compatible with cost-efficient oligo pool amplification. In A549 cells, the authors report an average of 3,749 decoded RNA molecules per cell from about 1,287 genes, with expression levels correlating with bulk RNA-seq and strong replicate reproducibility. The method generalizes to mouse tissues, mapping cell-type architectures and zonation programs in liver, placenta, and lymph node at single-molecule resolution. Finally, the Perturb-RAEFISH extension directly decodes gRNA spacer sequences in pooled CRISPR screens, detecting dozens of gRNA copies per cell and linking perturbations to spatial phenotypes without separate barcodes. Conclusion:RAEFISH expands spatial transcriptomics to genome-wide, single-molecule imaging and unlocks direct, image-based CRISPR perturbation readouts, setting the stage for unbiased discovery of spatial gene programs in development, physiology, and disease. Reference:Cheng Y, Dang S, Zhang Y, Chen Y, Yu R, Liu M, Jin S, Han A, Katz S, Wang S (2025). Sequencing-free whole-genome spatial transcriptomics at single-molecule resolution. Cell 188:1–18. https://doi.org/10.1016/j.cell.2025.09.006 License:This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International License (CC BY 4.0) – https://creativecommons.org/licenses/by/4.0/ Support:If you'd like to support Base by Base, you can make a one-time or monthly donation here: https://basebybase.castos.com/ Episode Slug: raefish-sequencing-free-whole-genome-spatial-transcriptomics Keywords: RAEFISH; spatial transcriptomics; single-molecule imaging; image-based CRISPR screen; liver zonation

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