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Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing
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Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing
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Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing
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Journal Article
Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing
2020
Overview
CRISPR–Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR–Cas9-mediated homology-independent organoid transgenesis (CRISPR–HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR–HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR–HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter—in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin—uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR–HOT simplifies genome editing in human organoids.Artegiani, Hendriks et al. describe a CRISPR–Cas9-based method to efficiently generate human knock-in organoids using non-homologous end joining to study rare intestinal cell types and human hepatocyte division.
