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Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening

Author

Listed:
  • Wei Jin

    (Heidelberg
    Parkville
    Parkville)

  • Yexuan Deng

    (Heidelberg
    Parkville
    Parkville
    Nanjing University)

  • John E. La Marca

    (Heidelberg
    Parkville
    Parkville)

  • Emily J. Lelliott

    (Heidelberg
    Parkville
    Parkville
    Bundoora)

  • Sarah T. Diepstraten

    (Parkville
    Parkville)

  • Christina König

    (Heidelberg
    Parkville
    Bundoora)

  • Lin Tai

    (Heidelberg)

  • Valentina Snetkova

    (South San Francisco)

  • Kristel M. Dorighi

    (South San Francisco)

  • Luke Hoberecht

    (South San Francisco)

  • Millicent G. Hedditch

    (Parkville)

  • Lauren Whelan

    (Parkville)

  • Geraldine Healey

    (Heidelberg)

  • Dan Fayle

    (Heidelberg)

  • Kieran Lau

    (Heidelberg
    Bundoora)

  • Margaret A. Potts

    (Heidelberg
    Parkville
    Parkville
    Bundoora)

  • Moore Z. Chen

    (Parkville)

  • Angus P. R. Johnston

    (Parkville)

  • Yang Liao

    (Heidelberg
    Bundoora)

  • Wei Shi

    (Heidelberg
    Bundoora)

  • Andrew J. Kueh

    (Heidelberg
    Parkville
    Parkville
    Bundoora)

  • Benjamin Haley

    (South San Francisco
    Centre de recherche de l’Hôpital Maisonneuve-Rosemont)

  • Jean-Philippe Fortin

    (South San Francisco)

  • Marco J. Herold

    (Heidelberg
    Parkville
    Parkville
    Bundoora)

Abstract

Cas12a is a next-generation gene editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.

Suggested Citation

  • Wei Jin & Yexuan Deng & John E. La Marca & Emily J. Lelliott & Sarah T. Diepstraten & Christina König & Lin Tai & Valentina Snetkova & Kristel M. Dorighi & Luke Hoberecht & Millicent G. Hedditch & Lau, 2025. "Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening," Nature Communications, Nature, vol. 16(1), pages 1-15, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-56282-2
    DOI: 10.1038/s41467-025-56282-2
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