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Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage
Authors:Yannik Bollen  Joris H. Hageman  Petra van Leenen  Lucca L. M. Derks  Bas Ponsioen  Julian R. Buissant des Amorie  Ingrid Verlaan-Klink  Myrna van den Bos  Leon W. M. M. Terstappen  Ruben van Boxtel  Hugo J. G. Snippert
Affiliation:1. Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, the Netherlands;2. Oncode Institute, Utrecht, the Netherlands;3. Medical Cell Biophysics, TechMed Centre, University of Twente, Enschede, the Netherlands;4. Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands; UNITED KINGDOM
Abstract:CRISPR-associated 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 non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. 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.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.
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