Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.

TitleGeneration of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.
Publication TypeJournal Article
Year of Publication2022
AuthorsLin, S-C, Haga, K, Zeng, X-L, Estes, MK
JournalNat Protoc
Volume17
Issue4
Pagination1004-1027
Date Published2022 Apr
ISSN1750-2799
KeywordsClone Cells, Cloning, Molecular, CRISPR-Cas Systems, Gene Editing, Gene Knockout Techniques, Humans, Lentivirus, RNA, Guide, Kinetoplastida
Abstract

Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR-Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR-Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2-small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2-3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.

DOI10.1038/s41596-021-00669-0
Alternate JournalNat Protoc
PubMed ID35197604
PubMed Central IDPMC9059808
Grant ListPO1AI057788 / / U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID) /
U19 AI144297 / AI / NIAID NIH HHS / United States
P42ES0327725 / / U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS) /
U19 AI116497 / AI / NIAID NIH HHS / United States
P30 ES030285 / ES / NIEHS NIH HHS / United States
HHSN2722017000381 / / U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID) /
CA125123 / / U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI) /
P01 AI057788 / AI / NIAID NIH HHS / United States
RP160283 / / Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas) /
P42 ES027725 / ES / NIEHS NIH HHS / United States
P30 DK056338 / DK / NIDDK NIH HHS / United States
U19AI144297 / / U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID) /
P30DK56338 / / U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases) /
P30ES030285 / / U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS) /
U19AI116497 / / U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID) /
RP17005 / / Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas) /