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

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)


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
Pages (from-to)e3001527
JournalPLoS biology
Issue number1
Publication statusPublished - Jan 2022


  • Alleles
  • Base Sequence
  • CRISPR-Associated Protein 9/genetics
  • Colon/cytology
  • DNA/genetics
  • DNA End-Joining Repair
  • Deoxyribonuclease I/genetics
  • Electroporation/methods
  • Epithelial Cells/cytology
  • Fluorescent Dyes/chemistry
  • Gene Knock-In Techniques
  • Genetic Loci
  • Genetic Vectors
  • Genome, Human
  • Heterozygote
  • Humans
  • Organoids/cytology
  • Recombinational DNA Repair
  • Staining and Labeling/methods


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