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INTESTINAL Publication-derived

Colorectal Cancer Colonic Organoids with Single-Cell Profiling

Full SOP with Academic or a plan

Source Ringel et al., 2020 · Institute of Molecular Health Sciences, ETH Zurich, and Department of Pharmacology and Toxicology, University of Zurich · 10.1016/j.stem.2020.02.007

Authors: Till Ringel, Nina Frey, Femke Ringnalda, Sharan Janjuha, Sarah Cherkaoui, Stefan Butz, Sumana Srivatsa, Martin Pirkl, Giancarlo Russo, Lukas Villiger, Gerhard Rogler, Hans Clevers, Niko Beerenwinkel, Nicola Zamboni, Tuncay Baubec, Gerald Schwank Duration: 30 days 8 phases Cell source: Human intestinal epithelial cells (derived from patient biopsies)

Abstract

This protocol describes an optimized method for genome-scale CRISPR screening in human small intestinal organoids using single-organoid sequencing analysis. The approach identifies genes controlling resistance to TGF-β-mediated growth restriction, including multiple tumor suppressors such as SWI/SNF chromatin remodeling complex subunits, enabling discovery of oncogenic signaling pathways relevant to colorectal cancer progression.

Cell source
Human intestinal epithelial cells (derived from patient biopsies)
Application
Disease modeling and driver gene identification via genome-wide CRISPR screening

Protocol overview

34 steps across 8 phases

Phase 1 · Organoid Culture and Expansion Day 0-7
  1. a Isolation of human intestinal crypts from biopsy tissue
  2. b Liberate crypts by vigorous shaking
  3. c Wash and concentrate crypts
  4. d Plate crypts in Matrigel
  5. e Establish organoid culture with growth factor medium
  6. f Expand organoids to desired cell numbers
Phase 2 · Lentivirus Production and Library Preparation Day 1-4 (in parallel with organoid culture)
  1. a Seed HEK293T cells for virus production
  2. b Prepare transfection mix
  3. c Transfect HEK293T cells
  4. d Harvest and concentrate lentivirus
  5. e Titrate lentivirus
Phase 3 · Organoid Dissociation and Lentiviral Transduction Day 7
  1. a Harvest and dissociate organoids to single cells
  2. b Stop enzymatic dissociation and wash
  3. c Perform spinoculation
  4. d Seed transduced cells in Matrigel
Phase 4 · Selection and Single Organoid Isolation Day 10-21
  1. a Apply selection pressure (day 7 post-transduction)
  2. b Allow organoids to grow under selection (1-2 weeks)
  3. c Pick single organoid clones
Phase 5 · Genomic DNA Extraction and sgRNA Amplification from Single Organoids Day 21-22
  1. a Extract genomic DNA from single organoids
  2. b PCR amplification of integrated sgRNAs
  3. c Purify PCR products
Phase 6 · Deep Sequencing and Data Processing Day 22-23
  1. a Prepare libraries for deep sequencing
  2. b Perform paired-end deep sequencing
  3. c Demultiplex reads by organoid barcode
Phase 7 · Hit Validation (Arrayed Rescreen) Day 1-7 (after hit identification)
  1. a Design and clone individual sgRNAs for validation
  2. b Produce lentivirus for individual sgRNAs
  3. c Transduce organoids with individual sgRNAs
  4. d Apply phenotypic selection and quantify outgrowth
  5. e Verify hits by alternative sgRNAs
Phase 8 · Optional: Confirmatory Analyses (Molecular and Functional) Day 1-30 (in parallel with other phases)
  1. a Confirm genetic modifications by deep sequencing
  2. b Assess SMAD2 phosphorylation by Western blot
  3. c Perform RNA-seq to assess transcriptional response
  4. d Analyze chromatin accessibility by ATAC-seq
  5. e Map transcription factor binding by CUT&RUN

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Attribution

This SOP was written by Organthis based on the published method in Ringel et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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