Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing

TY - JOUR

T1 - Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing

AU - Artegiani, Benedetta

AU - Hendriks, Delilah

AU - Beumer, Joep

AU - Kok, Rutger

AU - Zheng, Xuan

AU - Joore, Indi

AU - Chuva de Sousa Lopes, Susana

AU - van Zon, Jeroen

AU - Tans, Sander

AU - Clevers, Hans

PY - 2020/3

Y1 - 2020/3

N2 - 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.

AB - 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.

KW - CRISPR-Cas Systems

KW - Gene Editing

KW - Gene Knock-In Techniques/methods

KW - Hepatocytes/cytology

KW - Humans

KW - Intestines/cytology

KW - Liver/cytology

KW - Organoids/cytology

KW - Spindle Apparatus/ultrastructure

KW - Tumor Suppressor Protein p53/physiology

U2 - 10.1038/s41556-020-0472-5

DO - 10.1038/s41556-020-0472-5

M3 - Article

C2 - 32123335

SN - 1465-7392

VL - 22

SP - 321

EP - 331

JO - Nature Cell Biology

JF - Nature Cell Biology

IS - 3

ER -