Efficient CRISPR-Cas9-Mediated Generation of Knockin Human Pluripotent Stem Cells Lacking Undesired Mutations at the Targeted Locus
Open accessAbstract
The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.
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Reprogramming and genetic engineering have been implicated in increasing genomic variation.
“To further complicate this issue, reprogramming and genetic engineering itself have also been implicated in increasing genomic variation.169–171”
From Developments in stem cell-derived islet replacement therapy for treating type 1 diabetes (2023) · cites this paper as [171]
The Introduction reports that CRISPR-Cas9 engineering can produce off-target indels, and the Results describe undesired indels at the targeted locus in engineered human pluripotent stem cells. These passages support the genetic-engineering part of the claim, but our copy does not link reprogramming itself to increased genomic variation. (LLM, conf=0.87)
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- Authors
- Merkle, F. T., Neuhausser, W. M., Santos, D., Valen, E., Gagnon, J. A., Maas, K., Sandoe, J., Schier, A. F., & Eggan, K.
- Affiliations
- Broad Institute (US)
- Beth Israel Deaconess Medical Center (US)
- Howard Hughes Medical Institute (US)
- Harvard University (US)
- Corresponding author
- Eggan, Kevin
- ORCID
- Venue
- Cell Reports
- Publisher
- Elsevier BV
- Published
- 2015-05
- Volume
- 11
- Issue
- 6
- Pages
- 875-883
- ISSN
- 2211-1247 (ISSN-L)
- PMC
- PMC5533178
- OpenAlex
- W2162603028
- Cited by
- 152
- References
- 49
- Citations by year
- 2026: 3 · 2025: 4 · 2024: 9 · 2023: 13 · 2022: 15 · 2021: 8 · 2020: 15 · 2019: 14 · 2018: 21 · 2017: 21 · 2016: 25 · 2015: 4
- FWCI
- 10.28
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Cell Reports (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- NORA - Norwegian Open Research Archives (submittedVersion, cc-by-nc-nd)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- edoc (University of Basel) (submittedVersion)
- Apollo (University of Cambridge) (submittedVersion, cc-by-nc-nd)
- Funders
- Howard Hughes Medical Institute
- Bergens Forskningsstiftelse
- National Institutes of Health
- National Heart, Lung, and Blood Institute (R01HL109525, HL109525)
- National Institute of General Medical Sciences (P01GM099117)
- National Institute of Neurological Disorders and Stroke (R21NS071598, K99NS083713)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Keywords
- CRISPR/Cas9, human pluripotent stem cells, indel mutations, off-target mutations, Cas9 nickase, genome editing
- MeSH terms
- Base Sequence, Clone Cells, Humans, Molecular Sequence Data, Mutation, Gene Targeting, Pluripotent Stem Cells, INDEL Mutation, Gene Knock-In Techniques, Genetic Loci, CRISPR-Cas Systems
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