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

Marianna Paulis; Alessandra Castelli; Michela Lizier; Lucia Susani; Franco Lucchini; Anna Villa; Paolo Vezzoni

doi:10.1038/srep12327

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

Marianna Paulis, Alessandra Castelli, Michela Lizier, Lucia Susani, Franco Lucchini, Anna Villa, Paolo Vezzoni

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Abstract

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.

Original language	English
Article number	12327
Journal	Scientific Reports
Volume	5
DOIs	https://doi.org/10.1038/srep12327
Publication status	Published - Jul 24 2015

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

abstract = "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.",

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T1 - A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse embryonic stem cells

AU - Paulis, Marianna

AU - Castelli, Alessandra

AU - Lizier, Michela

AU - Susani, Lucia

AU - Lucchini, Franco

AU - Villa, Anna

AU - Vezzoni, Paolo

PY - 2015/7/24

Y1 - 2015/7/24

N2 - 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.

AB - 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.

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

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