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INTESTINAL Publication-derived

Sequential introduction of CRC mutations in cultured human intestinal stem cells using CRISPR/Cas9

Source Drost et al., 2015 · Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht · 10.1038/nature14415

👤 Jarno Drost, Richard H. van Jaarsveld, Bas Ponsioen, Cheryl Zimberlin, Ruben van Boxtel, Arjan Buijs, Norman Sachs, René M. Overmeer, G. Johan Offerhaus, Harry Begthel, Jeroen Korving, Marc van de Wetering, Gerald Schwank, Meike Logtenberg, Edwin Cuppen, Hugo J. Snippert, Jan Paul Medema, Geert J. P. L. Kops, Hans Clevers ⏱ 56 days 📋 9 phases 🧫 Human iPSC-derived intestinal stem cells from duodenal biopsies and colon tissue

Abstract

This protocol describes the sequential introduction of four colorectal cancer mutations (APC, P53, KRAS G12D, and SMAD4) into cultured human intestinal stem cell organoids using CRISPR/Cas9 genome editing. Mutant organoids are selected using functional selection strategies and can grow independently of stem-cell-niche factors. Upon xenotransplantation, quadruple mutants develop into tumors with invasive carcinoma features.

Cell source
Human iPSC-derived intestinal stem cells from duodenal biopsies and colon tissue
Application
Disease modeling - Colorectal cancer progression

Protocol overview

52 steps across 9 phases

Human organoid culture establishment and maintenance Days 1-ongoing
  1. 1 Obtain and prepare endoscopic duodenal biopsy samples
  2. 2 Prepare organoid culture medium
  3. 3 Culture organoids in complete WENR medium
APC knockout introduction and selection Days 3-14
  1. 1 Prepare organoid cells for transfection
  2. 2 Prepare Lipofectamine 2000 transfection complexes
  3. 3 Transfect organoid cells with APC sgRNA and Cas9
  4. 4 Replace medium with selection medium
  5. 5 Isolate and expand clonal APC knockout organoids
  6. 6 Verify APC knockout by genotyping
  7. 7 Confirm APC loss by qRT-PCR
P53 knockout introduction into APC knockout organoids Days 3-14 (relative to APC/P53 transfection)
  1. 1 Prepare APC knockout organoid cells for P53 transfection
  2. 2 Prepare and apply Lipofectamine 2000-P53 sgRNA/Cas9 complexes
  3. 3 Replace medium with nutlin-3 selection medium
  4. 4 Isolate and expand clonal APC/P53 double knockout organoids
  5. 5 Verify P53 knockout by genotyping
  6. 6 Confirm P53 loss by Western blot
KRAS G12D introduction into wild-type organoids Days 3-14
  1. 1 Design and prepare KRAS G12D homology-directed repair template
  2. 2 Prepare wild-type organoid cells for KRAS transfection
  3. 3 Prepare and apply Lipofectamine 2000 complexes with KRAS oligonucleotide
  4. 4 Replace medium with KRAS selection medium
  5. 5 Isolate and expand clonal KRAS G12D organoids
  6. 6 Verify KRAS G12D mutation by genotyping
SMAD4 knockout introduction into KRAS/APC/P53 triple mutants Days 3-14
  1. 1 Prepare KRAS G12D organoid cells for SMAD4 transfection
  2. 2 Prepare and apply Lipofectamine 2000 complexes with SMAD4 sgRNA
  3. 3 Replace medium with noggin-free selection medium
  4. 4 Isolate and expand clonal SMAD4 knockout organoids
  5. 5 Verify SMAD4 knockout by genotyping
  6. 6 Confirm SMAD4 loss by Western blot
In vivo tumorigenicity assessment - xenotransplantation Days 1-56 (8 weeks post-injection)
  1. 1 Prepare mutant organoids for injection
  2. 2 Prepare cell-Matrigel suspension for injection
  3. 3 Inject cell-Matrigel suspensions into NOD scid gamma mice
  4. 4 Monitor mice for nodule formation
  5. 5 Harvest tumors and process for analysis
Histological analysis and immunostaining Days 1-ongoing
  1. 1 Fix and process tumor tissues
  2. 2 Prepare tissue sections on slides
  3. 3 Perform H&E staining for histopathological assessment
  4. 4 Perform human cytokeratin (hKRT) immunostaining
  5. 5 Perform Ki67 immunostaining for proliferation assessment
  6. 6 Analyze tumor histology and classify
Chromosomal stability and aneuploidy assessment Days 1-ongoing
  1. 1 Transduce organoids with H2B-mNeon fluorescent histone lentivirus
  2. 2 Prepare organoids for live-cell imaging
  3. 3 Perform live-cell imaging of chromosome segregation
  4. 4 Analyze mitotic errors in live-cell imaging data
  5. 5 Prepare organoids for karyotyping
  6. 6 Perform metaphase chromosome spreading and analysis
  7. 7 Determine chromosome counts and identify aneuploidies
Off-target effect assessment Days 1-ongoing
  1. 1 Identify candidate off-target sites bioinformatically
  2. 2 Design amplicon primers for off-target sites
  3. 3 Perform PCR amplification of off-target candidates
  4. 4 Prepare DNA libraries and perform next-generation sequencing
  5. 5 Map sequences and identify indels
  6. 6 Evaluate off-target effects

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Drost et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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