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Review
. 2021 Oct;53(10):1483-1494.
doi: 10.1038/s12276-021-00609-8. Epub 2021 Oct 18.

Strategies for genetic manipulation of adult stem cell-derived organoids

Constantin Menche  1   2 , Henner F Farin  3   4   5   6
Affiliations
Review

Strategies for genetic manipulation of adult stem cell-derived organoids

Constantin Menche et al. Exp Mol Med. 2021 Oct.

Abstract

Organoid technology allows the expansion of primary epithelial cells from normal and diseased tissues, providing a unique model for human (patho)biology. In a three-dimensional environment, adult stem cells self-organize and differentiate to gain tissue-specific features. Accessibility to genetic manipulation enables the investigation of the molecular mechanisms underlying cell fate regulation, cell differentiation and cell interactions. In recent years, powerful methodologies using lentiviral transgenesis, CRISPR/Cas9 gene editing, and single-cell readouts have been developed to study gene function and carry out genetic screens in organoids. However, the multicellularity and dynamic nature of stem cell-derived organoids also present challenges for genetic experimentation. In this review, we focus on adult gastrointestinal organoids and summarize the state-of-the-art protocols for successful transgenesis. We provide an outlook on emerging genetic techniques that could further increase the applicability of organoids and enhance the potential of organoid-based techniques to deepen our understanding of gene function in tissue biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Organoids as a genetic model system.
Organoid lines derived from the gastrointestinal epithelium can be generated from isolated crypts or single adult stem cells (red). Ex vivo, stem cells self-renew and generate differentiated progeny (gray). By targeting single stem cells, transgenic organoids that can be clonally expanded and cryopreserved (green) are derived. In contrast, differentiated cells cannot form new organoids.
Fig. 2
Fig. 2. Stem cell dynamics in multicellular organoids.
Self-renewal in organoids is driven by stem cells that multiply as the organoids grow. Independent stem cells and their progeny are labeled in different colors. The clonal composition of organoids after passaging depends on the procedure: culture after single-cell dissociation and expansion of single organoids (“picking”) allows the derivation of clonal lines, whereas mechanical dissociation preserves larger fragments that contain multiple stem cells, resulting in mosaics. During repeated passaging, the organoids progressively become monoclonal due to neutral drift.
Fig. 3
Fig. 3. Strategies for the development of genetically modified organoids.
Top: Episomal transgene delivery, e.g., by transfection or electroporation of DNA plasmids, only allows transient expression, and organoids show progressive loss of transgene expression. Middle: Stable integration of transgenes, e.g., by lentiviral transduction combined with antibiotic selection, results in a population with homogenous expression. Right: Microscopic image showing the expression of green and red fluorescent lentiviral reporters after selection. Note that most organoids have a single color, indicating clonal outgrowth. Scale bar, 1 mm. Bottom: CRISPR/Cas9 technology allows targeting specific genomic loci. Correctly modified organoid lines can be obtained by the expansion of individual clones and subsequent genotyping.
Fig. 4
Fig. 4. Considerations for successful modification of gene function in organoids.
Flowchart depicting key questions that determine which strategies are most effective to obtain a desired transgenic organoid line. A factor is the anticipated phenotype of the gene of interest. Positively selectable traits such as growth advantages or discernable morphologic changes can be used to identify clonal organoids after NHEJ-mediated gene loss (a). For modifications that result in neutral or unknown phenotypes, a pre-enrichment strategy (e.g., by FACS) should be considered (b). If a defined alteration rather than gene loss is desired, classical HDR-mediated knock-in is recommended (c). To address phenotypes that result in compromised growth, inducible strategies, e.g., insertion of a conditional transcriptional termination sequence (d) or by knockdown using Tet-regulated lentiviral shRNA (e), are required. For additional information on the different approaches (a–e), please refer to Table 1.
Fig. 5
Fig. 5. Recent experimental approaches using CRISPR/Cas9 in organoids.
a Unbiased genetic screening in organoids after transduction with lentiviral gRNA libraries. After selection (e.g., for a growth advantage), the gRNA barcode distribution is analyzed by next-generation sequencing in a pooled organoid population or in individual organoid clones to identify genetic mediators. b Time-resolved lineage tracing using CRISPR/Cas9 techniques. The principle is based on the NHEJ-mediated introduction of “genetic scars” (asterisks) at neutral genomic positions that can be used to infer the phylogenetic history of cells.
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