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Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus

Merkle, Florian T. and Neuhausser, Werner M. and Santos, David and Valen, Eivind and Gagnon, James A. and Maas, Kristi and Sandoe, Jackson and Schier, Alexander F. and Eggan, Kevin. (2015) Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell Reports , 11 (6). pp. 875-883.

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Official URL: https://edoc.unibas.ch/74128/

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Abstract

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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Cell and Developmental Biology (Schier)
UniBasel Contributors:Schier, Alexander
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Elsevier
ISSN:2211-1247
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:24 May 2020 19:26
Deposited On:24 May 2020 19:26

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