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Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

Courtney M Mazur, Parthena E Kotsalidis, Majd George

    Biorxiv : the Preprint Server for Biology
    |November 24, 2025
    PubMed

    Insights

    Researchers identified CD61 as a marker for osteocyte maturation. A genome-wide screen revealed Clip2 is essential for osteocyte function, impacting CD61 expression and dendrite development.

    Area of Science:

    • Bone Biology
    • Cellular and Molecular Biology
    • Genomics

    Background:

    • Osteocytes are crucial for bone health, but the genes regulating their maturation and function remain largely unknown.
    • Understanding these genes is vital for developing therapeutics to enhance bone mass and strength.

    Purpose of the Study:

    • To identify novel genes involved in osteocyte differentiation and dendrite development.
    • To establish CD61 as a reliable marker for osteocyte maturation.

    Main Methods:

    • Genome-wide CRISPR-interference (CRISPRi) screening in an osteocyte-like cell line (Ocy454).
    • Development of a flow cytometry assay to quantify surface CD61 expression.
    • Analysis of gene expression and dendrite morphology following gene inhibition.

    Main Results:

    • CD61 (integrin β3) was identified as a marker of osteocyte maturation, with higher surface expression correlating with mature osteocyte markers.
    • A genome-wide screen identified Clip2, a microtubule-binding protein gene, as necessary for CD61 expression.
    • Clip2 inhibition reduced CD61 levels, decreased expression of osteocyte genes (Dmp1, Sost), and impaired dendrite morphology.

    Conclusions:

    • Surface CD61 is a valuable marker for assessing osteocyte maturity.
    • The microtubule cytoskeleton, influenced by Clip2, plays a significant role in osteocyte differentiation, morphology, and function.

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