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bioRxiv · 10.1101/2022.06.17.496589

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

Aguilar, G.·Bauer, M.·Vigano, M. A.·Jimenez-Jimenez, C.·Guerrero, I.·Affolter, M.
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Abstract

Precise genome engineering is essential for both basic and applied research. CRISPR/Cas accelerated the speed and ease by which defined exogenous sequences are integrated into specific loci. 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 describe SEED/Harvest, an efficient knock-in method based on homology-directed (HDR) and single-strand annealing (SSA) repair pathways. HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatio-temporal control of protein labelling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knock-ins, we have developed a toolbox based on rational engineering and functionalization of the ALFA nanobody.

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Aguilar, G., Bauer, M., Vigano, M. A., Jimenez-Jimenez, C., Guerrero, I., Affolter, M.. 2022-06-18. In vivo seamless genetic engineering via CRISPR-triggered single-strand annealing. https://doi.org/10.1101/2022.06.17.496589

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