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

Benedetta Artegiani, Delilah Hendriks, Joep Beumer, Rutger Kok, Xuan Zheng, Indi Joore, Susana Chuva de Sousa Lopes, Jeroen van Zon, Sander Tans, Hans Clevers

Research output: Contribution to journal/periodicalArticleScientificpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)321-331
Number of pages11
JournalNature Cell Biology
Volume22
Issue number3
DOIs
Publication statusPublished - Mar 2020

Keywords

  • CRISPR-Cas Systems
  • Gene Editing
  • Gene Knock-In Techniques/methods
  • Hepatocytes/cytology
  • Humans
  • Intestines/cytology
  • Liver/cytology
  • Organoids/cytology
  • Spindle Apparatus/ultrastructure
  • Tumor Suppressor Protein p53/physiology

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