PMID- 26204993
OWN - NLM
STAT- MEDLINE
DCOM- 20160802
LR  - 20240104
IS  - 2045-2322 (Electronic)
IS  - 2045-2322 (Linking)
VI  - 5
DP  - 2015 Jul 24
TI  - A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse 
      embryonic stem cells.
PG  - 12327
LID - 10.1038/srep12327 [doi]
LID - 12327
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.
FAU - Paulis, Marianna
AU  - Paulis M
AD  - 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale 
      delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, 
      Milan, Italy.
FAU - Castelli, Alessandra
AU  - Castelli A
AD  - 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale 
      delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, 
      Milan, Italy.
FAU - Lizier, Michela
AU  - Lizier M
AD  - 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale 
      delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, 
      Milan, Italy.
FAU - Susani, Lucia
AU  - Susani L
AD  - 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale 
      delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, 
      Milan, Italy.
FAU - Lucchini, Franco
AU  - Lucchini F
AD  - Catholic University of the Sacred Heart, Cremona, Italy.
FAU - Villa, Anna
AU  - Villa A
AD  - 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale 
      delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, 
      Milan, Italy.
FAU - Vezzoni, Paolo
AU  - Vezzoni P
AD  - 1] Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale 
      delle Ricerche, Milan, Italy [2] Humanitas Clinical and Research Center, Rozzano, 
      Milan, Italy.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20150724
PL  - England
TA  - Sci Rep
JT  - Scientific reports
JID - 101563288
RN  - EC 2.4.2.8 (Hypoxanthine Phosphoribosyltransferase)
SB  - IM
MH  - Animals
MH  - *CRISPR-Cas Systems
MH  - Hypoxanthine Phosphoribosyltransferase/genetics/metabolism
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Mice
MH  - Mouse Embryonic Stem Cells/cytology/*metabolism
PMC - PMC4513284
EDAT- 2015/07/25 06:00
MHDA- 2016/08/03 06:00
PMCR- 2015/07/24
CRDT- 2015/07/25 06:00
PHST- 2014/11/03 00:00 [received]
PHST- 2015/05/22 00:00 [accepted]
PHST- 2015/07/25 06:00 [entrez]
PHST- 2015/07/25 06:00 [pubmed]
PHST- 2016/08/03 06:00 [medline]
PHST- 2015/07/24 00:00 [pmc-release]
AID - srep12327 [pii]
AID - 10.1038/srep12327 [doi]
PST - epublish
SO  - Sci Rep. 2015 Jul 24;5:12327. doi: 10.1038/srep12327.