Publication: Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System
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Identifiers
Date
2020-06-18
Authors
Sánchez-Hernández, Sabina
Aguilar-González, Araceli
Guijarro-Albaladejo, Beatriz
Maldonado-Pérez, Noelia
Ramos-Hernández, Iris
Cortijo-Gutiérrez, Marina
Sánchez Martín, Rosario María
Benabdellah, Karim
Martin, Francisco
Advisors
Journal Title
Journal ISSN
Volume Title
Publisher
MDPI
Abstract
In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.
Description
MeSH Terms
Medical Subject Headings::Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA::DNA, Recombinant
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Gene Targeting
Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome Components::Genes::Genes, Reporter
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Genetic Engineering
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Therapeutics::Biological Therapy::Genetic Therapy
Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genetic Vectors
Medical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans
Medical Subject Headings::Anatomy::Cells::Cells, Cultured::Cell Line::Cell Line, Tumor::K562 Cells
Medical Subject Headings::Organisms::Viruses::RNA Viruses::Retroviridae::Lentivirus
Medical Subject Headings::Chemicals and Drugs::Macromolecular Substances::Polymers::Biopolymers::Microfilament Proteins::Wiskott-Aldrich Syndrome Protein Family::Wiskott-Aldrich Syndrome Protein
Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Processes::Gene Expression Regulation::Epigenesis, Genetic::Gene Silencing::CRISPR-Cas Systems
Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Processes::Recombination, Genetic::Homologous Recombination
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Models, Theoretical::Models, Biological::Models, Genetic
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Gene Targeting
Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome Components::Genes::Genes, Reporter
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Genetic Engineering
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Therapeutics::Biological Therapy::Genetic Therapy
Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genetic Vectors
Medical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans
Medical Subject Headings::Anatomy::Cells::Cells, Cultured::Cell Line::Cell Line, Tumor::K562 Cells
Medical Subject Headings::Organisms::Viruses::RNA Viruses::Retroviridae::Lentivirus
Medical Subject Headings::Chemicals and Drugs::Macromolecular Substances::Polymers::Biopolymers::Microfilament Proteins::Wiskott-Aldrich Syndrome Protein Family::Wiskott-Aldrich Syndrome Protein
Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Processes::Gene Expression Regulation::Epigenesis, Genetic::Gene Silencing::CRISPR-Cas Systems
Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Processes::Recombination, Genetic::Homologous Recombination
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Models, Theoretical::Models, Biological::Models, Genetic
DeCS Terms
CIE Terms
Keywords
Models, DNA donor, On-target integration, Homologous directed recombination (HDR), Efficacy, Safety, Specificity, eGFP, dsRED, CRISPR/Cas9, Modelos estructurales, Reparación del ADN por recombinación, Eficacia, Seguridad, Especificidad, Proteínas fluorescentes verdes, Proteína 9 asociada a CRISPR, ADN recombinante
Citation
Sánchez-Hernández S, Aguilar-González A, Guijarro-Albaladejo B, Maldonado-Pérez N, Ramos-Hernández I, Cortijo-Gutiérrez M, et al.Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System. Cells. 2020 Jun 18;9(6):1492