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.
Original language | English |
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Pages (from-to) | e3001527 |
Journal | PLoS biology |
Volume | 20 |
Issue number | 1 |
DOIs | |
Publication status | Published - Jan 2022 |
Keywords
- 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