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

• Published in: PLoS biology 2022-01, Vol.20 (1), p.e3001527-e3001527
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Alleles; Base Sequence; Biology and Life Sciences; Clonal selection; Cloning; Colon - cytology; Colon - metabolism; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; Deoxyribonuclease I - genetics; Deoxyribonuclease I - metabolism; Deoxyribonucleic acid; DNA; DNA - genetics; DNA - metabolism; DNA damage; DNA End-Joining Repair; Earth Sciences; Editing; Efficiency; Electroporation - methods; Epithelial Cells - cytology; Epithelial Cells - metabolism; Experiments; Fluorescence; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Gene Knock-In Techniques; Genetic Loci; Genetic Vectors; Genome editing; Genome, Human; Genomes; Genomics; Heterozygote; Homology; Humans; Localization; Marking; Medicine and Health Sciences; Methods and Resources; Mutation; Nicking endonuclease; Non-homologous end joining; Nuclease; Organoids; Organoids - cytology; Organoids - metabolism; Physical Sciences; Physiological aspects; Recombinational DNA Repair; Research and Analysis Methods; Staining and Labeling - methods; Tagging
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.3001527

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.