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

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 nonhomologous 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.

Applications of human organoids in the personalized treatment for digestive diseases

“… 139 , 512 – 514 The distribution and function of target genes during digestive development or progression of disease can be detected in real time by gene knockin fluorescent labeling in iPSCs. 514 , 515 Single-cell RNA sequencing (scRNA-seq) methods have changed the paradigm of biomedical science by analyzing cellular heterogeneity from multivariate data. 516 scRNA-seq of organoids has allowed certain rare or abnormal cell types to be identified, and these findings can be detected in individual organoids.…”

RNF43 mutations facilitate colorectal cancer metastasis via formation of a tumour-intrinsic niche

“…CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 23, 2022. ; https://doi.org/10.1101/2022.12.22.521159 doi: bioRxiv preprint cloning 54 . iCaspase-T2A-tdTomato sequence was removed and swapped for mNeonGreen-NLS using a combination of Q5® High-Fidelity DNA Polymerase and the In-Fusion HD Cloning kit (Takara).…”

3D imaging for driving cancer discovery

“…Genetically encoded reporters for live 3D imaging have been used to extend the application of live imaging to visualization of key processes in cancer biology (Hirata et al , 2015; Kondo et al , 2021; Ponsioen et al , 2021). Development of novel reporters (Mahlandt et al , 2021), multiplexing approaches that combine several reporters (Regot et al , 2014; Chavez‐Abiega et al , 2022) and improved genome editing technologies (Bollen et al , 2022) can be expected to further expand fluorescent reporter applications and increase the richness of information that can be extracted from live 3D imaging data.…”