Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage
Bollen, Yannik; Hageman, Joris H; van Leenen, Petra; Derks, Lucca L M; Ponsioen, Bas; Buissant des Amorie, Julian R; Verlaan-Klink, Ingrid; van den Bos, Myrna; Terstappen, Leon W M M; van Boxtel, Ruben; Snippert, Hugo J G
(2022) PLoS Biology, volume 20, issue 1, pp. 1 - 16
(Article)
Abstract
AU CRISPR-associated: Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly nucleases are powerful tools for precise : genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or nonhomologous end joining (NHEJ)-mediated integration of the desired knock-in.
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A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.
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Keywords: General Agricultural and Biological Sciences, General Biochemistry,Genetics and Molecular Biology, General Immunology and Microbiology, General Neuroscience, Journal Article
ISSN: 1544-9173
Publisher: Public Library of Science
Note: Funding Information: This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. HGJS received European Research Council (ERC) starting grant (IntratumoralNiche), project number 803608 (https://erc.europa.eu/funding/starting-grants) and NWO TOP. YB was supported by a strategic alliance between University of Twente and UMC Utrecht on Advanced Biomanufacturing (to LWMMT and HJGS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank members of the Snippert laboratory for reagents, suggestions, and discussions. We thank Markus J. van Roosmalen for advice on the WGS analysis. Publisher Copyright: Copyright: © 2022 Bollen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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