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INTESTINAL Publication-derived

Colorectal Cancer Colonic Organoids

Full SOP with Academic or a plan

Source Michels et al., 2020 · Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany · 10.1016/j.stem.2020.04.003

Authors: Birgitta E. Michels, Mohammed H. Mosa, Barbara I. Streibl, Tianzuo Zhan, Constantin Menche, Khalil Abou-El-Ardat, Tahmineh Darvishi, Ewelina Cz1onka, Sebastian Wagner, Jan Winter, Hind Medyouf, Michael Boutros, Henner F. Farin Duration: 84 days 7 phases Cell source: Human colon organoids (normal)

Abstract

This protocol establishes a platform for pooled CRISPR-Cas9 screening in human colon organoids to identify tumor suppressors in vitro and after xenotransplantation in vivo. The method uses lentiviral CRISPR-Cas9 libraries with unique molecular identifiers (UMIs) to enable clonal tracing and distinguish clone size and abundance, allowing unbiased identification of genes conferring positive selection in normal and pre-malignant organoids.

Cell source
Human colon organoids (normal)
Application
Disease modeling; Tumor suppressor gene identification

Protocol overview

38 steps across 7 phases

Phase 1 · Organoid Culture and Cas9 Expression Days 1-14
  1. a Establish organoid culture
  2. b Transduce organoids with lentiCas9-Blast
  3. c Select Cas9-expressing organoids
Phase 2 · TGF-β Pathway Validation Screen (Training Library) Days 15-56
  1. a Prepare and titer training library lentivirus
  2. b Transduce organoids with training library
  3. c Collect T0 control
  4. d Establish no-selection control (NSC)
  5. e Initiate TGF-β selection
  6. f Harvest selected organoids
Phase 3 · Pre-Malignant Organoid Generation (APC/KRAS/TP53) Days 1-60
  1. a Introduce APC knockout
  2. b Introduce KRAS G12D mutation
  3. c Introduce TGFBR2 knockout (AKT line)
  4. d Characterize AK organoid stability
Phase 4 · In Vivo TSG Library Screening in Xenografted Organoids Days 1-84
  1. a Prepare and titer TSG library lentivirus
  2. b Transduce AK organoids with TSG library
  3. c Collect T0 control
  4. d Establish NSC and prepare for transplantation
  5. e Prepare organoids for transplantation
  6. f Subcutaneous transplantation into NSG mice
  7. g Monitor tumor growth
  8. h Harvest tumors and extract genomic DNA
Phase 5 · CRISPR-UMI Validation Screen with Clonal Tracing Days 1-56
  1. a Construct CRISPR-UMI validation library
  2. b Titer and transduce AK organoids with UMI library
  3. c Collect T0 and NSC controls
  4. d Prepare organoids for transplantation
  5. e Subcutaneous transplantation and tumor growth
  6. f Harvest tumors and extract genomic DNA
Phase 6 · Barcode Amplification and Next-Generation Sequencing Days 1-7
  1. a Amplify training library barcodes
  2. b Amplify TSG library barcodes (two-step PCR)
  3. c Amplify UMI library barcodes
  4. d Prepare libraries for sequencing
  5. e Perform next-generation sequencing
Phase 7 · Bioinformatic Analysis and gRNA Enrichment Days 1-14
  1. a Demultiplex and parse barcode sequences
  2. b Normalize read counts and calculate gRNA enrichment
  3. c Identify enriched gRNAs and gene hits
  4. d Analyze UMI library data for clonal abundance and incidence
  5. e Perform gene set enrichment analysis (GSEA)
  6. f Statistical testing and multiple comparison correction

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Attribution

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

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