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  3. A microscopy-based CRISPR screening platform enables organellar functional genomics and illuminates ciliary biology

A microscopy-based CRISPR screening platform enables organellar functional genomics and illuminates ciliary biology

  • Dev Cell. 2025 Nov 20:S1534-5807(25)00666-5. doi: 10.1016/j.devcel.2025.10.015.
Jingbo Sun 1,  Irem Sude Atiş 1,  Stéfany L L Empke 2,  Mustafa K Khokha 2,  David K Breslow 3
Affiliations

Affiliations

  • 1 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA.
  • 2 Department of Pediatrics and Department of Genetics, Yale University School of Medicine, New Haven, CT, USA; Department of Pediatrics, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
  • 3 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA; Wu Tsai Institute, Yale University, New Haven, CT, USA. Electronic address: [email protected].
PMID: 41270742 DOI: 10.1016/j.devcel.2025.10.015
Abstract

Microscopy offers an indispensable technique for visualizing biological processes and for defining cytological abnormalities characteristic of disease. However, combining microscopy with the power of pooled CRISPR screening presents considerable technical challenges, hindering application of systematic genetic analysis to imaging-defined phenotypes. Here, we establish a fluorescence microscopy-based CRISPR screening platform that combines ease of implementation with flexible analysis of live-cell or antibody-based molecular markers, including post-translational modifications. Applying this methodology, we systematically identify regulators of primary cilium structure and function in human cells through targeted and genome-wide screens. We further show that integration of screens focused on distinct ciliary phenotypes yields multi-dimensional profiles that delineate precise gene functions. Among the identified hits, TZMP1 (SMIM27) encodes a microprotein at the ciliary transition zone that is required for ciliogenesis in human cells and for ciliary function in Xenopus embryos. More broadly, our approach provides a technological and conceptual strategy for microscopy-based functional genomics.

Keywords

CRISPR screen; axoneme; cilia; ciliopathy; functional genomics; microprotein; optical screening; polyglutamylation; transition zone.

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