Skip to content
← Back to browse
INTESTINAL Publication-derived

Sequential Cancer Mutations in Cultured Human Intestinal Stem Cells via 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 ⏱ 70 days 📋 8 phases 🧫 Human small intestinal and colon stem cells from endoscopic biopsies

Abstract

This protocol describes the sequential introduction of four colorectal cancer driver mutations (APC, P53, KRAS, SMAD4) into cultured human intestinal stem cell organoids using CRISPR/Cas9 technology with functional selection strategies. Mutant organoids progress from niche-dependent to niche-independent growth in vitro and form invasive carcinomas upon xenotransplantation into mice.

Cell source
Human small intestinal and colon stem cells from endoscopic biopsies
Application
Disease modeling; colorectal cancer progression

Protocol overview

56 steps across 8 phases

Organoid Culture Establishment and Maintenance Days 1–14
  1. 1 Obtain human tissue samples
  2. 2 Prepare organoid culture medium
  3. 3 Establish and expand wild-type organoid cultures
APC Inactivation via CRISPR/Cas9 Days 3–14
  1. 1 Prepare organoids for transfection
  2. 2 Prepare lipofection complexes for APC targeting
  3. 3 Transfect organoid cultures
  4. 4 Apply functional selection for APC loss
  5. 5 Isolate and expand clonal APC knockout organoids
  6. 6 Genotype APC knockout clones
  7. 7 Verify APC knockout by qRT-PCR
P53 Inactivation in APC[KO] Organoids Days 3–14
  1. 1 Prepare APC[KO] organoids for transfection
  2. 2 Prepare lipofection complexes for P53 targeting
  3. 3 Transfect and culture
  4. 4 Apply nutlin-3 selection for P53 inactivation
  5. 5 Isolate and expand clonal APC[KO]/P53[KO] organoids
  6. 6 Genotype P53 knockout clones
  7. 7 Verify P53 knockout by western blot and qRT-PCR
KRAS[G12D] Introduction in Wild-Type Organoids Days 3–14
  1. 1 Prepare wild-type organoids for transfection
  2. 2 Prepare homology-directed repair template oligonucleotide
  3. 3 Prepare lipofection complexes for KRAS[G12D] homology-directed repair
  4. 4 Transfect organoid cultures
  5. 5 Apply EGFR inhibitor selection for KRAS[G12D]
  6. 6 Isolate and expand clonal KRAS[G12D] organoids
  7. 7 Genotype KRAS[G12D] clones
Introduction of Multi-Gene Mutation Combinations (KRAS[G12D]/APC[KO]/P53[KO]/SMAD4[KO]) Days 3–14
  1. 1 Prepare KRAS[G12D] organoids for APC sgRNA transfection
  2. 2 Introduce APC knockout into KRAS[G12D] organoids
  3. 3 Apply dual selection for KRAS[G12D]/APC[KO]
  4. 4 Isolate and expand KRAS[G12D]/APC[KO] clones
  5. 5 Introduce P53 knockout into KRAS[G12D]/APC[KO] organoids
  6. 6 Apply triple selection with nutlin-3
  7. 7 Isolate and expand KRAS[G12D]/APC[KO]/P53[KO] triple-mutant clones
  8. 8 Introduce SMAD4 knockout into triple-mutant organoids
  9. 9 Apply quadruple selection (all niche factors withdrawn)
  10. 10 Isolate and expand clonal quadruple-mutant organoids
  11. 11 Genotype all four loci in quadruple-mutant clones
  12. 12 Verify quadruple-mutant phenotype by western blot
In Vivo Tumorigenicity Assessment via Xenotransplantation Days 1–56
  1. 1 Expand organoids in selection medium
  2. 2 Harvest organoid cells
  3. 3 Prepare Matrigel suspension
  4. 4 Prepare NSG mice
  5. 5 Perform subcutaneous injection
  6. 6 Monitor mice for tumor growth
  7. 7 Euthanize mice and harvest tumors
  8. 8 Fix and process tissues for histology
Histological and Immunohistochemical Analysis Days 50–70
  1. 1 Perform hematoxylin and eosin (H&E) staining
  2. 2 Perform human cytokeratin immunostaining
  3. 3 Perform Ki67 proliferation staining
  4. 4 Document and interpret results
Chromosome Stability and Aneuploidy Analysis Days 1–21 (Live-cell imaging); Days 1–3 (Karyotyping)
  1. 1 Prepare organoids for live-cell imaging
  2. 2 Plate H2B-mNeon organoids for imaging
  3. 3 Perform live-cell mitosis imaging
  4. 4 Analyze mitotic errors from live-cell imaging
  5. 5 Prepare organoids for karyotyping
  6. 6 Prepare metaphase spreads
  7. 7 Stain chromosomes and acquire images
  8. 8 Document representative karyotypes

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

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.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence