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INTESTINAL Publication-derived

CRISPR-Cas9–Mediated Engineering of Human Intestinal Organoids for Colorectal Cancer Modeling

Source Matano et al., 2015 · Keio University School of Medicine, Tokyo, Japan · 10.1038/nm.3802

👤 Mami Matano, Shoichi Date, Mariko Shimokawa, Ai Takano, Masayuki Fujii, Yuki Ohta, Toshiaki Watanabe, Takanori Kanai, Toshiro Sato ⏱ 240 days 📋 9 phases 🧫 Normal human intestinal epithelial cells from endoscopic biopsy samples

Abstract

This protocol details the stepwise introduction of recurrent driver pathway mutations (APC, KRAS[G12V], SMAD4, TP53, PIK3CA[E545K]) into human intestinal organoids using CRISPR-Cas9 genome editing combined with niche factor-modulated selection. The engineered organoids progressively lose dependence on exogenous niche factors and acquire tumorigenic capacity proportional to the number of mutations, enabling prospective analysis of human colorectal carcinogenesis mechanisms.

Cell source
Normal human intestinal epithelial cells from endoscopic biopsy samples
Application
Disease modeling - colorectal cancer progression via sequential driver mutations

Protocol overview

51 steps across 9 phases

Organoid dissociation and APC targeting Day 0–3
  1. 1 Dissociate organoids into single cells
  2. 2 Electroporate APC-targeting CRISPR-Cas9 vectors
  3. 3 Culture electroporated cells and select for APC mutations
  4. 4 Validate APC mutations by SURVEYOR assay and sequencing
  5. 5 Verify β-catenin upregulation by Western blot
SMAD4 and TP53 targeting Day 4–10
  1. 1 Dissociate normal organoids and target SMAD4
  2. 2 Culture and select SMAD4-targeted organoids
  3. 3 Validate SMAD4 mutations and loss of protein
  4. 4 Dissociate normal organoids and target TP53
  5. 5 Select TP53-targeted organoids using nutlin-3
  6. 6 Validate TP53 mutations and overexpression
KRAS G12V knock-in and selection Day 11–20
  1. 1 Dissociate A-organoids and prepare for KRAS G12V knock-in
  2. 2 Co-electroporate KRAS G12V sgRNA and donor vector
  3. 3 Culture cells in EGF-free medium to enrich KRAS G12V mutants
  4. 4 Validate KRAS G12V knock-in by Southern blot and sequencing
  5. 5 Verify constitutive KRAS activation by phospho-ERK Western blot
PIK3CA E545K knock-in in KRAS[G12V] organoids Day 21–30
  1. 1 Dissociate KRAS[G12V] A-organoids for PIK3CA targeting
  2. 2 Co-electroporate PIK3CA E545K sgRNA and donor oligonucleotide
  3. 3 Select PIK3CA E545K mutants in EGF-free, MEK inhibitor-containing medium
  4. 4 Validate PIK3CA E545K knock-in by sequencing
  5. 5 Verify PIK3CA activation by phospho-AKT Western blot
Generation of multi-mutant organoids (AKSTP) and clonal expansion Day 31–60
  1. 1 Progressively introduce mutations through sequential rounds of gene editing
  2. 2 Generate AKSTP organoids by targeting PIK3CA in AKST organoids
  3. 3 Validate all five mutations in AKSTP organoids by whole-genome sequencing
  4. 4 Perform copy number and karyotype analysis
  5. 5 Perform whole-mount immunohistochemistry on AKSTP organoids
Gene expression profiling and transcriptional analysis Day 61–75
  1. 1 Extract RNA from engineered, adenoma, and CRC organoids
  2. 2 Perform microarray hybridization
  3. 3 Analyze gene expression data and perform GSEA
  4. 4 Perform principal component analysis (PCA)
  5. 5 Identify differentially expressed genes between engineered and CRC organoids
  6. 6 Submit microarray data to GEO database
Xenotransplantation into kidney subcapsule of NOG mice Day 76–120
  1. 1 Prepare GFP-labeled organoids for transplantation
  2. 2 Prepare organoids for implantation
  3. 3 Implant organoids under kidney subcapsule of NOG mice
  4. 4 Monitor tumor growth at 1 and 2 months post-transplantation
  5. 5 Perform histological analysis of xenografts
  6. 6 Perform in situ hybridization for LGR5 mRNA in xenografts
  7. 7 Culture recovered tumor organoids in vitro
Splenic injection and assessment of metastatic potential Day 121–180
  1. 1 Prepare GFP-labeled organoids for splenic injection
  2. 2 Inject organoid cells into mouse spleen
  3. 3 Monitor for liver metastases at 2 months post-injection
  4. 4 Prepare metastatic tissue for histological analysis
  5. 5 Recover cells from AKSTP micrometastases for retransplantation
Engineering adenoma organoids and assessment of metastatic capacity Day 181–240
  1. 1 Establish adenoma-derived organoid lines from human adenoma tissue
  2. 2 Engineer adenoma organoids with TP53 and SMAD4 mutations
  3. 3 Introduce KRAS G12V or PIK3CA E545K into Ade[CIN] TS organoids
  4. 4 Perform morphological assessment of engineered adenoma organoids in vitro
  5. 5 Perform copy number analysis on engineered adenoma organoids
  6. 6 Transplant Ade[CIN] TSK/TSP organoids into kidney subcapsule
  7. 7 Assess metastatic capacity by splenic injection

Full SOP

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Attribution

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

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