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

• Published in: Nature communications 2025-01, Vol.16 (1), p.974-15, Article 974
• Main Authors: Jin, Wei, Deng, Yexuan, La Marca, John E., Lelliott, Emily J., Diepstraten, Sarah T., König, Christina, Tai, Lin, Snetkova, Valentina, Dorighi, Kristel M., Hoberecht, Luke, Hedditch, Millicent G., Whelan, Lauren, Healey, Geraldine, Fayle, Dan, Lau, Kieran, Potts, Margaret A., Chen, Moore Z., Johnston, Angus P. R., Liao, Yang, Shi, Wei, Kueh, Andrew J., Haley, Benjamin, Fortin, Jean-Philippe, Herold, Marco J.
• Format: Article
• Language: English
• Subjects: 13; 49; 631/1647/1511; 631/208/4041/3196; 631/67/70; 64/60; Animals; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cell activation; CRISPR; CRISPR-Associated Proteins - genetics; CRISPR-Associated Proteins - metabolism; CRISPR-Cas Systems - genetics; Editing; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Fibroblasts; Fibroblasts - metabolism; Fluorescence; Gene Editing - methods; Gene Knock-In Techniques - methods; Gene Knockout Techniques - methods; Gene targeting; Genetic engineering; Genetic Engineering - methods; Genetic modification; Genome editing; Genomes; Humanities and Social Sciences; Humans; In vitro methods and tests; In vivo methods and tests; Lymphocytes; Lymphocytes T; Lymphoma; Lymphoma - genetics; Mice; Mice, Inbred C57BL; multidisciplinary; Multiplexing; Red Fluorescent Protein; Science; Science (multidisciplinary); Stem cells; Transformed cells; Transgenic mice
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-025-56282-2

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. Cas12a represents the next generation of gene editing. Here, the authors present the generation and validation of a Cas12a transgenic mouse model. Additionally, the authors create whole-genome Cas12a knockout libraries, and demonstrate their utility across multiple in vitro and in vivo screens.