In vivo seamless genetic engineering via CRISPR-triggered single-strand annealing

Aguilar, Gustavo and Bauer, Milena and Vigano, M. Alessandra and Jiménez-Jiménez, Carlos and Guerrero, Isabel and Affolter, Markus. (2022) In vivo seamless genetic engineering via CRISPR-triggered single-strand annealing.

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Official URL: https://edoc.unibas.ch/88618/

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Precise genome engineering is essential for both basic and applied research, permitting the manipulation of genes and gene products in predictable ways. The irruption of the CRISPR/Cas technology accelerated the speed and ease by which defined exogenous sequences are integrated into specific loci. To this day, a number of strategies permit gene manipulation. Nevertheless, knock-in generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Even when achieved, the analysis of protein localization can still be unfeasible in highly packed tissues, where spatial and temporal control of gene labeling would be ideal. Here, we propose an efficient method based on homology-directed repair (HDR) and single-strand annealing (SSA) repair pathways. In this method, HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. By engineering the Hedgehog (Hh) pathway components, we demonstrated fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. The use of homology arms as short as 30 base pairs further simplified and cheapened the process. In addition, SSA can be triggered in somatic cells, permitting conditional gene labeling in different tissues. Finally, to achieve conditional labeling and manipulation of proteins tagged with short protein tags, we have further developed a toolbox based on rational engineering and functionalization of the ALFA nanobody.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Cell Biology (Affolter)
UniBasel Contributors:Affolter, Markus
Item Type:Preprint
Note:Publication type according to Uni Basel Research Database: Discussion paper / Internet publication
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Last Modified:21 Jul 2022 03:10
Deposited On:19 Jul 2022 10:42

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