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INTESTINAL Publication-derived

Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids

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

👤 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 ⏱ 84 days 📋 7 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Michels et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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