read_status: unread
type: paper
title: "An organoid-based CRISPR-Cas9 screen for regulators of intestinal epithelial maturation and cell fate"
year: 2023
authors:
- "Hansen, Stine L."
- "Larsen, Hjalte L."
- "Pikkupeura, Laura M."
- "Maciag, Grzegorz"
- "Guiu, Jordi"
- "Müller, Iris"
- "Clement, Ditte L."
- "Mueller, Christina"
- "Johansen, Jens Vilstrup"
- "Helin, Kristian"
- "Lerdrup, Mads"
- "Jensen, Kim B."
journal: "Science Advances"
doi: "10.1126/sciadv.adg4055"
url: "https://www.science.org/doi/10.1126/sciadv.adg4055"
project:
- EpPathobio
system:
- intestinal_epithelium
- intestinal_organoids
- fetal_intestinal_progenitors
- adult_intestinal_epithelium
disease:
- intestinal_injury
- inflammatory_bowel_disease
genes:
- smarca4
- smarcc1
- yap1
processes:
- epithelial_maturation
- cell_fate_transition
- chromatin_remodeling
- transcriptional_repression
- tuft_cell_differentiation
methods:
- crispr_cas9_screen
- intestinal_organoid_culture
- scrna_seq
- atac_seq
- in_vivo_transplantation
key_findings:
- fetal_state_maintenance
- epigenetic_barrier_to_maturation
- yap1_program_suppression
- adult_like_cell_state_induction
limitations:
- incomplete_in_vitro_maturation
- partial_lineage_specification
- organoid_context_dependence
relevance:
- regulators_of_epithelial_maturation
- epigenetic_control_of_cell_identity
- organoid_screening_strategy
concepts:
- epigenetic_barriers_to_differentiation
- organoid_based_genetic_screens
- yap_dependent_fetal_programs
- chromatin_remodeling_in_cell_fate
tags:
- gi_epithelium
- development
- chromatin
- organoids
Generation of functionally mature organs requires exquisite control of transcriptional programs governing cell state transitions during development. Despite advances in understanding the behavior of adult intestinal stem cells and their progeny, the transcriptional regulators that control the emergence of the mature intestinal phenotype remain largely unknown. Using mouse fetal and adult small intestinal organoids, we uncover transcriptional differences between the fetal and adult state and identify rare adult-like cells present in fetal organoids. This suggests that fetal organoids have an inherent potential to mature, which is locked by a regulatory program. By implementing a CRISPR-Cas9 screen targeting transcriptional regulators expressed in fetal organoids, we establish Smarca4 and Smarcc1 as important factors safeguarding the immature progenitor state. Our approach demonstrates the utility of organoid models in the identification of factors regulating cell fate and state transitions during tissue maturation and reveals that SMARCA4 and SMARCC1 prevent precocious differentiation during intestinal development. Functional perturbation screen in intestinal organoids uncovers that SWI/SNF components safeguard tissue maturation.
Fetal intestinal organoids harbor rare adult-like cells, indicating latent maturation capacity.
CRISPR-Cas9 loss-of-function screening identifies SMARCA4 and SMARCC1 as repressors of epithelial maturation.
Disruption of SMARCA4 or SMARCC1 reduces fetal progenitor markers and increases adult secretory and tuft-like cells.
Loss of SMARCA4/SMARCC1 shifts chromatin accessibility toward an adult-like epigenetic landscape.
SMARCA4/SMARCC1 maintain a YAP1-driven fetal transcriptional program that blocks maturation.
Targeted CRISPR-Cas9 pooled screen in fetal intestinal organoids.
Flow cytometry–based sorting using SCA1 and CD117 as maturation markers.
Single-cell RNA-seq to resolve cell-state transitions.
ATAC-seq to assess chromatin accessibility changes.
In vivo transplantation into adult mouse colon to test maturation competence.
Establishes epigenetic repression, not lack of competence, as a key barrier to fetal-to-adult transition.
Positions SWI/SNF chromatin remodelers as gatekeepers of developmental timing.
Demonstrates feasibility of organoid-based genetic screens for developmental regulators.
Highlights YAP1 activity as a central node coupling mechanics, chromatin, and cell fate.
Figure 1: scRNA-seq reveals adult-like clusters within fetal organoids.
Figure 2: Design and validation of CRISPR-Cas9 screen in fetal organoids.
Figure 3: SMARCA4/SMARCC1 disruption drives loss of fetal markers.
Figure 4: Mutant organoids show enhanced adult intestinal identity after transplantation.
Figure 5: Transcriptomic and chromatin remodeling toward adult state.
Figure 6: Increased frequency of adult-like tuft cells upon SMARCA4/SMARCC1 loss.
Genetic disruption does not fully recapitulate complete adult epithelial maturation in vitro.
Lineage output biased toward tuft cells, not full secretory spectrum.
Organoid culture conditions may constrain differentiation trajectories.
SWI/SNF complex composition dynamics not directly dissected.