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INTESTINAL Publication-derived

Sequential Cancer Mutations in Cultured Human Intestinal Stem Cells - CRISPR/Cas9-Mediated Genome Editing

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 ⏱ 196 days 📋 8 phases 🧫 Human intestinal stem cells (normal duodenal and colon tissue)

Abstract

This protocol establishes a method for sequential introduction of four colorectal cancer mutations (APC, P53, KRAS[G12D], and SMAD4) into cultured human intestinal organoids using CRISPR/Cas9 technology. Mutant organoids are selected using functional assays based on growth factor dependence, and quadruple mutants form invasive carcinomas upon xenotransplantation.

Cell source
Human intestinal stem cells (normal duodenal and colon tissue)
Application
Disease modeling - Colorectal cancer progression

Protocol overview

52 steps across 8 phases

Organoid Culture Establishment and Preparation Days 1-7
  1. 1 Obtain human intestinal tissue
  2. 2 Establish organoid culture
  3. 3 Expand organoids in complete growth medium
APC Knockout Introduction and Selection Days 4-21
  1. 1 Prepare organoids for transfection
  2. 2 Prepare Cas9 and APC sgRNA complexes
  3. 3 Transfect organoids
  4. 4 Plate transfected cells in matrix
  5. 5 Apply functional selection for APC loss
  6. 6 Isolate and expand clonal organoids
  7. 7 Genotype APC[KO] organoids
  8. 8 Confirm APC[KO] pathway activity via qRT-PCR
P53 Knockout Introduction and Selection in APC[KO] Background Days 22-39
  1. 1 Prepare APC[KO] organoids for P53 transfection
  2. 2 Prepare Cas9 and P53 sgRNA complexes
  3. 3 Transfect APC[KO] organoids with P53 sgRNA
  4. 4 Plate transfected cells in matrix
  5. 5 Apply nutlin-3 selection for P53 loss
  6. 6 Isolate and expand clonal P53[KO]/APC[KO] organoids
  7. 7 Genotype P53[KO] organoids
  8. 8 Confirm P53 loss via Western blot and qRT-PCR
KRAS[G12D] Introduction via Homologous Recombination Days 40-70
  1. 1 Design and synthesize KRAS[G12D] targeting oligonucleotide
  2. 2 Prepare wild-type organoids for KRAS transfection
  3. 3 Prepare Cas9, KRAS sgRNA, and oligonucleotide complexes
  4. 4 Transfect organoids with KRAS components
  5. 5 Plate transfected cells in matrix
  6. 6 Apply EGF withdrawal and gefitinib selection
  7. 7 Isolate and expand clonal KRAS[G12D] organoids
  8. 8 Genotype KRAS[G12D] organoids
Simultaneous Introduction of APC, P53, and SMAD4 Knockouts in KRAS[G12D] Background Days 71-105
  1. 1 Prepare KRAS[G12D] organoids for multiplex transfection
  2. 2 Prepare Cas9, APC, P53, and SMAD4 sgRNA complex for triple mutation
  3. 3 Transfect KRAS[G12D] organoids with triple sgRNA mix
  4. 4 Plate transfected cells in matrix
  5. 5 Apply sequential functional selection for triple mutants
  6. 6 Isolate and expand clonal triple-mutant organoids
  7. 7 Genotype triple-mutant organoids
  8. 8 Confirm triple-mutant protein expression via Western blot
Generation of Complete Quadruple Mutants (APC/P53/KRAS/SMAD4) Days 106-140
  1. 1 Prepare triple-mutant organoids for additional selection
  2. 2 Apply complete niche factor withdrawal (noggin removal)
  3. 3 Isolate and expand quadruple-mutant organoids
  4. 4 Genotype quadruple-mutant organoids
  5. 5 Confirm quadruple-mutant phenotype in minimal medium
In Vivo Tumorigenicity Assessment Days 141-196
  1. 1 Prepare organoid cell suspensions for transplantation
  2. 2 Mix organoid cells with Matrigel
  3. 3 Perform subcutaneous xenotransplantation
  4. 4 Monitor mice for tumor development
  5. 5 Harvest tumors and process for histology
  6. 6 Perform histological analysis
Chromosomal Instability and Aneuploidy Assessment Days 21-70 (parallel to mutation introduction)
  1. 1 Transduce organoids with H2B fluorescent tag
  2. 2 Perform live-cell imaging of mitosis
  3. 3 Score mitotic errors manually
  4. 4 Perform karyotyping
  5. 5 Count chromosomes and quantify aneuploidy
  6. 6 Generate representative karyograms

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.

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