Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

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*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

7 Citations (Scopus)
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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.
Original languageEnglish
Article numbere3001527
Pages (from-to)1-16
JournalPLoS Biology
Issue number1
Publication statusPublished - 28 Jan 2022


  • Organoids
  • CRISPR/Cas9
  • Genome editing
  • fluorescent protein
  • live cell microscopic imaging


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