Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening
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 gen...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-01-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-56282-2 |
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| author | 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 Marco J. Herold |
| author_facet | 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 Marco J. Herold |
| author_sort | Wei Jin |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-2b913446578449d4819602520c489f35 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-2b913446578449d4819602520c489f352025-02-02T12:31:35ZengNature PortfolioNature Communications2041-17232025-01-0116111510.1038/s41467-025-56282-2Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screeningWei Jin0Yexuan Deng1John E. La Marca2Emily J. Lelliott3Sarah T. Diepstraten4Christina König5Lin Tai6Valentina Snetkova7Kristel M. Dorighi8Luke Hoberecht9Millicent G. Hedditch10Lauren Whelan11Geraldine Healey12Dan Fayle13Kieran Lau14Margaret A. Potts15Moore Z. Chen16Angus P. R. Johnston17Yang Liao18Wei Shi19Andrew J. Kueh20Benjamin Haley21Jean-Philippe Fortin22Marco J. Herold23Olivia Newton-John Cancer Research Institute, HeidelbergOlivia Newton-John Cancer Research Institute, HeidelbergOlivia Newton-John Cancer Research Institute, HeidelbergOlivia Newton-John Cancer Research Institute, HeidelbergThe Walter and Eliza Hall Institute of Medical Research, ParkvilleOlivia Newton-John Cancer Research Institute, HeidelbergOlivia Newton-John Cancer Research Institute, HeidelbergDepartment of Molecular Biology, Genentech, Inc., South San FranciscoDepartment of Molecular Biology, Genentech, Inc., South San FranciscoComputational Sciences, Genentech, Inc., South San FranciscoThe Walter and Eliza Hall Institute of Medical Research, ParkvilleThe Walter and Eliza Hall Institute of Medical Research, ParkvilleOlivia Newton-John Cancer Research Institute, HeidelbergOlivia Newton-John Cancer Research Institute, HeidelbergOlivia Newton-John Cancer Research Institute, HeidelbergOlivia Newton-John Cancer Research Institute, HeidelbergDrug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, ParkvilleDrug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, ParkvilleOlivia Newton-John Cancer Research Institute, HeidelbergOlivia Newton-John Cancer Research Institute, HeidelbergOlivia Newton-John Cancer Research Institute, HeidelbergDepartment of Molecular Biology, Genentech, Inc., South San FranciscoComputational Sciences, Genentech, Inc., South San FranciscoOlivia Newton-John Cancer Research Institute, HeidelbergAbstract 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.https://doi.org/10.1038/s41467-025-56282-2 |
| spellingShingle | 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 Marco J. Herold Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening Nature Communications |
| title | Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening |
| title_full | Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening |
| title_fullStr | Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening |
| title_full_unstemmed | Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening |
| title_short | Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening |
| title_sort | advancing the genetic engineering toolbox by combining ascas12a knock in mice with ultra compact screening |
| url | https://doi.org/10.1038/s41467-025-56282-2 |
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