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INTESTINAL Publication-derived

Modeling Colorectal Cancer Using Organoids from Familial APC

Source Drost et al. · Hubrecht Institute and University Medical Centre Utrecht, Utrecht, Netherlands · 10.1126/science.aao3130

👤 Drost J, van Boxtel R, Blokzijl F, Mizutani T, Sasaki N, Duursma A, de Ligt J, Offerhaus GJ, Begthel H, Korber J, van de Wetering M, Schwank G, Clevers H ⏱ 120 days 📋 6 phases 🧫 Patient-Derived (FAP Patient Normal and Adenoma Tissue)

Abstract

Used organoids from familial adenomatous polyposis patients to demonstrate that APC-heterozygous normal intestinal epithelium accumulates mutations at an accelerated rate compared to wild-type tissue. Whole-genome sequencing revealed distinct mutational signatures active during early tumorigenesis.

Cell source
Patient-Derived (FAP Patient Normal and Adenoma Tissue)
Application
Cancer Prevention

Protocol overview

25 steps across 6 phases

CRISPR-Cas9 Gene Targeting and Knockout Generation Days 1–14
  1. 1 Design and Prepare CRISPR Components
  2. 2 Transfect Organoids with CRISPR-Cas9 and Selection Cassette
  3. 3 Puromycin Selection
  4. 4 Clonal Expansion and Single Organoid Isolation
  5. 5 Genotyping by PCR and Sequencing
  6. 6 Verify Gene Inactivation by qRT-PCR
  7. 7 Confirm Protein Loss by Western Blot
Mutation Accumulation Period and Subclonal Derivation Days 15–90 (MLH1) or Days 15–120 (NTHL1)
  1. 8 Passage Knockout and Parental Organoids
  2. 9 Generate Subclonal Cultures via Flow Cytometry
  3. 10 Expand Subclonal Organoids
DNA Extraction and Whole-Genome Sequencing Days 91–120+
  1. 11 Isolate Genomic DNA
  2. 12 Perform Whole-Genome Sequencing (WGS)
  3. 13 Align Sequencing Reads and Call Variants
  4. 14 Filter and Annotate Mutations
  5. 15 Calculate Mutation Accumulation Rates
Mutational Signature Extraction and Analysis Post-sequencing
  1. 16 Extract Mutational Spectra
  2. 17 Compare to COSMIC Signatures
  3. 18 Analyze Indel Patterns
Replication Timing and Genomic Distribution Analysis Post-sequencing
  1. 19 Perform Replication Timing Analysis (Repli-seq)
  2. 20 Determine Replication Origin Locations
  3. 21 Correlate Mutations with Replication Timing
  4. 22 Analyze Replication Strand Asymmetry
Validation and Comparison to Clinical Samples Post-analysis
  1. 23 Obtain Clinical Cancer Sequencing Data
  2. 24 Perform Cosine Similarity Analysis on Clinical Samples
  3. 25 Screen for Germline Mutations in Candidate Genes

Full SOP

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Attribution

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

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