A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse embryonic stem cells

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated 9 (Cas9) technology has been recently added to the tools allowing efficient and easy DNA targeting, representing a very promising approach to gene engineering. Using the CRISPR/Cas9 system we have driven the integration...

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Bibliographic Details
Published in:Scientific reports 2015-07, Vol.5 (1), p.12327-12327, Article 12327
Main Authors: Paulis, Marianna, Castelli, Alessandra, Lizier, Michela, Susani, Lucia, Lucchini, Franco, Villa, Anna, Vezzoni, Paolo
Format: Article
Language:English
Subjects:
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14/32
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631/208/1405
631/532/2117
Animals
CRISPR
CRISPR-Cas Systems
Deoxyribonucleic acid
DNA
Embryo cells
Embryos
Fluorescence in situ hybridization
Humanities and Social Sciences
Hypoxanthine phosphoribosyltransferase
Hypoxanthine Phosphoribosyltransferase - genetics
Hypoxanthine Phosphoribosyltransferase - metabolism
In Situ Hybridization, Fluorescence - methods
Integration
Mice
Mouse Embryonic Stem Cells - cytology
Mouse Embryonic Stem Cells - metabolism
multidisciplinary
Nucleotide sequence
Science
Stem cell transplantation
Stem cells
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Summary:The clustered regularly interspaced short palindromic repeat (CRISPR)/associated 9 (Cas9) technology has been recently added to the tools allowing efficient and easy DNA targeting, representing a very promising approach to gene engineering. Using the CRISPR/Cas9 system we have driven the integration of exogenous DNA sequences to the X-linked Hprt gene of mouse embryonic stem cells. We show here that a simple fluorescence in situ hybridization (FISH)-based strategy allows the detection and the frequency evaluation of non-specific integrations of a given plasmid. FISH analysis revealed that these integrations do not match the software predicted off-target loci. We conclude that the frequency of these CRISPR-mediated off-target DNA cuts is negligible, since, due to the occurrence of spontaneous double-strand breaks, we observed more aspecific plasmid integrations than those corresponding to predicted off-target sites.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep12327