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.
authors.affiliation: 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
authors.forename: Marianna
authors.initials: M
authors.lastname: Paulis
authors.affiliation: 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Castelli
authors.affiliation: 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Lizier
authors.affiliation: 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Susani
authors.affiliation: Catholic University of the Sacred Heart, Cremona, Italy.
authors.forename: Franco
authors.initials: F
authors.lastname: Lucchini
authors.affiliation: 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Villa
authors.affiliation: 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Vezzoni
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D007041:Hypoxanthine Phosphoribosyltransferase