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

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, Lauren Whelan, Geraldine Healey, Dan Fayle, Kieran Lau, Margaret A. Potts, Moore Z. Chen, Angus P. R. Johnston, Yang Liao, Wei Shi, Andrew J. Kueh, Benjamin Haley, Jean-Philippe Fortin and Marco J. Herold ()
Additional contact information
Wei Jin: Heidelberg
Yexuan Deng: Heidelberg
John E. La Marca: Heidelberg
Emily J. Lelliott: Heidelberg
Sarah T. Diepstraten: Parkville
Christina König: Heidelberg
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
Margaret A. Potts: Heidelberg
Moore Z. Chen: Parkville
Angus P. R. Johnston: Parkville
Yang Liao: Heidelberg
Wei Shi: Heidelberg
Andrew J. Kueh: Heidelberg
Benjamin Haley: South San Francisco
Jean-Philippe Fortin: South San Francisco
Marco J. Herold: Heidelberg

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: 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.

Date: 2025
References: View references in EconPapers View complete reference list from CitEc
Citations:

Downloads: (external link)
https://www.nature.com/articles/s41467-025-56282-2 Abstract (text/html)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-56282-2

Ordering information: This journal article can be ordered from
https://www.nature.com/ncomms/

DOI: 10.1038/s41467-025-56282-2

Access Statistics for this article

Nature Communications is currently edited by Nathalie Le Bot, Enda Bergin and Fiona Gillespie

More articles in Nature Communications from Nature
Bibliographic data for series maintained by Sonal Shukla () and Springer Nature Abstracting and Indexing ().