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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MDPI
Materia
Models DNA donor On-target integration Homologous directed recombination (HDR) Efficacy Safety Specificity eGFP dsRED CRISPR/Cas9
Date
2020-06-18Referencia bibliográfica
Sánchez-Hernández, S., Aguilar-González, A., Guijarro-Albaladejo, B., Maldonado-Pérez, N., Ramos-Hernández, I., Cortijo-Gutiérrez, M., ... & Martin, F. (2020). Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System. Cells, 9(6), 1492. [doi: 10.3390/cells9061492]
Sponsorship
Spanish ISCIII Health Research Fund; European Union (EU) PI15/02015 PI18/00337 PI18/00330; CECEyU of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) 2016000073391-TRA 2016000073332-TRA PI-57069 CARTPI-0001-201 PAIDI-Bio326 PI-0014-2016; CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) 2016000073391-TRA 2016000073332-TRA PI-57069 CARTPI-0001-201 PAIDI-Bio326 PI-0014-2016; Nicolas Monardes regional Ministry of Health contract 0006/2018; Fundacion Poco Frecuente; Spanish Government FPU17/04327 FPU17/02268; ISCIII through a PFIS fellowship FI19/00163; SMSI PEJ-2018-001760-AAbstract
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.