Generation of Knockout Gene-Edited Human Intestinal Organoids

Chathruckan Rajendra; Tomas Wald; Kevin Barber; Jason R Spence; Faranak Fattahi; Ophir D Klein

doi:10.1007/978-1-0716-0747-3_13

Generation of Knockout Gene-Edited Human Intestinal Organoids

Methods Mol Biol. 2020:2171:215-230. doi: 10.1007/978-1-0716-0747-3_13.

Authors

Chathruckan Rajendra^{1

2}, Tomas Wald², Kevin Barber^{3

4}, Jason R Spence^{5

6

7

8}, Faranak Fattahi^{3

4}, Ophir D Klein^{9

10

11

12}

Affiliations

¹ Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA.
² Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, San Francisco, CA, USA.
³ Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA.
⁴ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA.
⁵ Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
⁶ Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.
⁷ Department of Biomedical Engineering, College of Engineering, University of Michigan, Ann Arbor, MI, USA.
⁸ Center for Organogenesis, University of Michigan Medical School, Ann Arbor, MI, USA.
⁹ Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA. ophir.klein@ucsf.edu.
¹⁰ Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, San Francisco, CA, USA. ophir.klein@ucsf.edu.
¹¹ Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. ophir.klein@ucsf.edu.
¹² Klein Lab, UCSF, San Francisco, CA, USA. ophir.klein@ucsf.edu.

Abstract

We discuss a methodology to generate and study knockout gene-edited human intestinal organoids. We describe the generation of knockout human embryonic stem cell lines that we then differentiate into mature human intestinal organoid tissue in Matrigel using several growth factors. We also discuss a pair of assays that can be used to study the integrity of the intestinal epithelial barrier of the human intestinal organoids under inflammatory stress conditions.

Keywords: Crispr-Cas9; Gene editing; Human embryonic stem cells; Human intestinal organoids; Transfection.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics
Cells, Cultured
Embryonic Stem Cells / cytology*
Embryonic Stem Cells / metabolism*
Gene Editing
Humans
Intestinal Mucosa / cytology*
Organoids / cytology*
Organoids / metabolism*
Transfection

Grants and funding

T32 DK007762/DK/NIDDK NIH HHS/United States