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INTESTINAL Publication-derived

Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients

Source Schwank et al., 2013 · Hubrecht Institute/KNAW and University Medical Center Utrecht · 10.1016/j.stem.2013.11.002

👤 Gerald Schwank, Bon-Kyoung Koo, Valentina Sasselli, Johanna F. Dekkers, Inha Heo, Turan Demircan, Nobuo Sasaki, Sander Boymans, Edwin Cuppen, Cornelis K. van der Ent, Edward E.S. Nieuwenhuis, Jeffrey M. Beekman, Hans Clevers ⏱ 30 days 📋 7 phases 🧫 Patient-Derived iPSC (Cystic Fibrosis)

Abstract

This protocol describes the use of CRISPR/Cas9 genome editing to correct the CFTR F508del mutation in patient-derived intestinal stem cell organoids. Corrected organoids are expanded clonally, validated by sequencing, and functionally assessed using a forskolin-induced swelling assay to demonstrate restoration of CFTR function.

Cell source
Patient-Derived iPSC (Cystic Fibrosis)
Application
Gene correction and disease modeling

Protocol overview

46 steps across 7 phases

Organoid Culture and Optimization (Mouse APC) 0–14 days
  1. 1 Culture intestinal organoids in Wnt-conditioned medium
  2. 2 Trypsinize organoids to obtain single cell suspension
  3. 3 Transfect cells with CRISPR/Cas9 plasmids targeting APC
  4. 4 Plate single transfected cells in medium without R-spondin
  5. 5 Expand organoids for two weeks
  6. 6 Isolate individual organoid clones and verify morphology
  7. 7 Sequence APC locus to confirm mutations
Human APC Editing Optimization 0–14 days
  1. 1 Culture human intestinal organoids
  2. 2 Trypsinize human organoids to single cell suspension
  3. 3 Transfect with CRISPR/Cas9 plasmids targeting human APC
  4. 4 Plate transfected cells in medium without Wnt and R-spondin
  5. 5 Expand organoid clones for two weeks
  6. 6 Select clones with cystic morphology and verify by sequencing
CF Patient Organoid Establishment and Characterization 0–30 days
  1. 1 Isolate intestinal tissue from CF patients
  2. 2 Establish SI and LI organoid cultures from patient tissue
  3. 3 Perform forskolin-induced swelling assay on wild-type control organoids
  4. 4 Perform forskolin-induced swelling assay on CF patient organoids
  5. 5 Document baseline phenotype by quantitative image analysis
CFTR F508del Correction by CRISPR/Cas9 0–21 days
  1. 1 Design two sgRNAs targeting CFTR F508del locus
  2. 2 Generate donor plasmid with CFTR wild-type sequences
  3. 3 Incorporate puromycin selection cassette in donor plasmid
  4. 4 Culture CF patient organoids in complete growth medium
  5. 5 Trypsinize CF organoids into single cell suspension
  6. 6 Cotransfect CF organoid single cells with Cas9/sgRNA and donor plasmid
  7. 7 Plate transfected single cells in selection medium
  8. 8 Expand puromycin-resistant organoid clones for 2 weeks
  9. 9 Isolate individual puromycin-resistant organoid clones
Validation of CFTR Correction by Genotyping 0–7 days
  1. 1 Extract genomic DNA from corrected organoid clones
  2. 2 Perform PCR with external primers to confirm donor plasmid integration
  3. 3 Sequence PCR products to confirm F508del correction
  4. 4 Determine zygosity of corrected alleles by genomic sequencing
  5. 5 Extract total RNA from corrected organoid clones
  6. 6 Perform RT-PCR with allele-specific primers
  7. 7 Sequence RT-PCR products to confirm corrected mRNA
Functional Validation of Corrected CFTR 0–1 day
  1. 1 Label organoids with calcein-green dye
  2. 2 Perform forskolin-induced swelling assay on corrected organoids
  3. 3 Quantify organoid swelling by automated image analysis
  4. 4 Treat corrected organoids with CFTR-specific inhibitor CFTRinh-172
  5. 5 Calculate area under the curve (AUC) for swelling kinetics
  6. 6 Compare functional restoration to wild-type and CF controls
Off-Target Analysis 0–7 days
  1. 1 Identify potential off-target CRISPR sites computationally
  2. 2 Select representative off-target sites for sequencing
  3. 3 Extract genomic DNA from one representative corrected clone per sgRNA
  4. 4 PCR amplify off-target sites from genomic DNA
  5. 5 Sequence off-target amplicons
  6. 6 Characterize any detected off-target mutations

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Schwank et al., 2013. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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