Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids
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Abstract
Prime editing is a recently reported genome editing tool using a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. Here, we explore the use of prime editing in human organoids. Common TP53 mutations can be correctly modeled in human adult stem cell–derived colonic organoids with efficiencies up to 25% and up to 97% in hepatocyte organoids. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9–mediated homology-directed repair and adenine base editing on the CFTR-R785* mutation. Whole-genome sequencing of prime editing–repaired organoids revealed no detectable off-target effects. Despite encountering varying editing efficiencies and undesired mutations at the target site, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.
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Contrary to base editing, efficient use of prime editing uses two sgRNAs that require significant optimization, resulting in need to test multiple sgRNA combinations.
“Contrary to base editing, efficient use of prime editing uses two sgRNAs that require significant optimization for efficient use. This results in the need to test multiple sgRNA combinations to find the most effective combination65,66.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [66]
The Introduction (page not available) describes a pegRNA and a second PE3 guide RNA that can improve prime-editing efficiency, and the Methods report using guide pairs. But the second guide is presented as an enhancement, not a requirement; neither those passages nor the Discussion’s call for further improvement says that both guides require significant optimization or that multiple combinations must be tested to find the best one. (LLM, conf=0.84)
Experimental — accuracy not yet measuredWe have previously used prime editing in hepatocyte and colon organoids to model tumorigenesis.
“We have previously used prime editing in hepatocyte and colon organoids to model tumorigenesis66.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [66]
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- Authors
- Geurts, M. H., de Poel, E., Pleguezuelos-Manzano, C., Oka, R., Carrillo, L., Andersson-Rolf, A., Boretto, M., Brunsveld, J. E., van Boxtel, R., Beekman, J. M., & Clevers, H.
- Affiliations
- Royal Netherlands Academy of Arts and Sciences (NL)
- Department of Pediatric Respiratory Medicine, Wilhelmina Children’s Hospital, University Medical Center, Utrecht University, Utrecht, the Netherlands
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center Utrecht, Utrecht, the Netherlands
- Oncode Institute (NL)
- Utrecht University (NL)
- Oncode Institute,Princes Maxima Center, Utrecht, The Netherlands
- Corresponding author
- Clevers, Hans
- ORCID
- Venue
- Life Science Alliance
- Publisher
- Life Science Alliance, LLC
- Published
- 2021-08-09
- Volume
- 4
- Issue
- 10
- Pages
- e202000940
- ISSN
- 2575-1077 (ISSN-L)
- PMC
- PMC8356249
- OpenAlex
- W3188144652
- Cited by
- 151
- References
- 47
- Citations by year
- 2026: 22 · 2025: 27 · 2024: 29 · 2023: 35 · 2022: 23 · 2021: 12 · 2020: 2
- FWCI
- 7.76
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Life Science Alliance (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- KNAW research portal (Royal Academy of Art and Sciences (KNAW)) (publishedVersion, other-oa)
- Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
- Funders
- Cancer Research UK (C6307/A29058)
- The Mark Foundation for Cancer Research
- NWO Building Blocks of Life Project: Cell Dynamics within Lung and Intestinal Organoids (737.016.009)
- Dutch Cystic Fibrosis Foundation (NCFS), HIT-CF Program, the Dutch Health Organization ZonMw, the Netherlands
- The Netherlands Organ-on-Chip Initiative, an NWO Gravitation Project (024.003.001)
- Ministry of Education, Culture and Science of the government of the Netherlands
- Oncode Institute, Dutch Cancer Society, Gravitation Program
- The Netherlands Organisation for Scientific Research
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- prime editing, CRISPR/Cas9 nickase, human organoids, colonic organoids, hepatocyte organoids, TP53 mutation, cancer modeling, cystic fibrosis, F508del mutation, adenine base editing, homology-directed repair, off-target effects, genome editing
- MeSH terms
- Gene Editing, Alleles, Animals, Epithelial Cells, Humans, Mutation, Oncogenes, Organoids, Genetic Therapy, Sequence Analysis, DNA, Cystic Fibrosis Transmembrane Conductance Regulator, Amino Acid Substitution, Hepatocytes, CRISPR-Cas Systems
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