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
Olivia Newton-John Cancer Research Institute, Heidelberg, Melbourne, Australia.
The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Australia.
Nat Commun. 2025 Jan 30;16(1):974. 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是一种能够实现多重基因靶向的新一代基因编辑工具。在此，我们展示了一种组成性表达与mCherry荧光报告基因相连的增强型嗜酸嗜热栖热菌Cas12a（enAsCas12a）的小鼠模型。我们利用来自enAsCas12a小鼠的原代细胞和转化细胞，在体外证明了高效的单基因和多基因编辑。我们进一步利用正常的和易患癌症的enAsCas12a干细胞重建野生型小鼠的造血系统，证明了体内基因编辑的成功。我们还展示了紧凑的全基因组Cas12a敲除文库，每个基因在一个（《谐谑曲》）或两个（《小步舞曲》）载体上编码四个crRNA，并证明了这些文库在多种方法中的实用性：分别在淋巴瘤细胞和永生化成纤维细胞中进行体外富集和缺失筛选，以及在体内筛选以鉴定驱动淋巴瘤的事件。最后，我们利用原代T细胞和成纤维细胞通过同时进行基因敲除（通过Cas12a）和激活（通过dCas9-SAM）证明了CRISPR多重编辑。我们的enAsCas12a小鼠和配套的crRNA文库增强了基因组工程能力，并补充了当前的CRISPR技术。
