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Cited by 110CRISPR and Genetic EngineeringPluripotent Stem Cells ResearchRNA regulation and disease

Genome Editing in Human Pluripotent Stem Cells: Approaches, Pitfalls, and Solutions

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Hendriks, William T., Warren, Curtis R., Cowan, Chad A.·2016·Cell Stem Cell
DOI 10.1016/j.stem.2015.12.002PMC PMC4709030
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Abstract

Human pluripotent stem cells (hPSCs) with knockout or mutant alleles can be generated using custom-engineered nucleases. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nucleases are the most commonly employed technologies for editing hPSC genomes. In this Protocol Review, we provide a brief overview of custom-engineered nucleases in the context of gene editing in hPSCs with a focus on the application of TALENs and CRISPR/Cas9. We will highlight the advantages and disadvantages of each method and discuss theoretical and technical considerations for experimental design.

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  • CRISPR-Cas9 impacts on quality include an undefined level of off-target effects of designer nucleases, purity of the Cas-9 protein, constitutive expression of Cas-9, genetic stability of edited clones, and retention of wild-type cells.

    “As an example of the most commonly used technique, CRISPR-Cas9 impacts on quality include: an undefined level of off-target effects of designer nucleases, purity of the Cas-9 protein, constitutive expression of Cas-9, genetic stability of edited clones, and retention of wild-type cells (Hendriks et al., 2016).”

    From Guidance document on Good cell and tissue culture practice 2.0 (GCCP 2.0) (2021) · cites this paper as Hendriks et al., 2016

    The sections on CRISPR guide design and the Concluding remarks identify off-target cutting as a concern and say sustained Cas9 expression can exacerbate nuclease activity. The section on genome-edited cell selection warns that merged colonies can produce mixed genotypes, while the paper also discusses mutations accumulated during cell culture. Our copy does not identify Cas9 protein purity or specifically the retention of wild-type cells as quality concerns, and it does not characterize off-target effects as having an undefined level. Page numbers are unavailable in our copy. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Three general strategies are being investigated: use of immunosuppressive or immunomodulatory drugs; use of a physical barrier (e.g., encapsulation); and gene editing for immune evasion and/or immune protection.

    “Three general strategies are being investigated: 1) use of immunosuppressive or immunomodulatory drugs; 2) use of a physical barrier (e.g., encapsulation) (288); and 3) gene editing for immune evasion and/or immune protection (289).”

    From The management of type 1 diabetes in adults. A consensus report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) (2021) · cites this paper as [289]

    Citation 289 is attached to gene editing for immune evasion or protection, not to the drug or encapsulation strategies. The Introduction describes genome editing in human pluripotent stem cells for disease modeling, but the passages found for immune evasion, immune protection, transplantation rejection, and HLA do not address editing cells to avoid immune responses. Our copy therefore does not verify the cited point. (LLM, conf=0.72)

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Cited by

Sorted by citations

Authors
Hendriks, W. T., Warren, C. R., & Cowan, C. A.
Affiliations
Harvard University (US)
Corresponding author
Cowan, Chad A.
ORCID
Warren, Curtis R.
Venue
Cell Stem Cell
Publisher
Elsevier BV
Published
2016-01
Volume
18
Issue
1
Pages
53-65
ISSN
  • 1875-9777 (ISSN-L)
  • 1934-5909
DOI
10.1016/j.stem.2015.12.002
PMC
PMC4709030
OpenAlex
W2227959865
Cited by
110
References
14
Citations by year
2026: 3 · 2025: 6 · 2024: 3 · 2023: 6 · 2022: 8 · 2021: 10 · 2020: 13 · 2019: 12 · 2018: 12 · 2017: 16 · 2016: 21
FWCI
1.84
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell stem cell (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Heart, Lung, and Blood Institute (U01HL100408, U01HL107440)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK095384, R01DK097768)
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 › Molecular Biology › RNA regulation and disease
Keywords
genome editing, human pluripotent stem cells, TALEN, CRISPR/Cas9, engineered nucleases, knockout alleles, genome editing efficiency, experimental design
MeSH terms
Alleles, Deoxyribonucleases, DNA, Genetic Techniques, Genetic Vectors, Genotype, Humans, Mutation, Sequence Homology, Nucleic Acid, Genome, Human, Point Mutation, Pluripotent Stem Cells, CRISPR-Cas Systems

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