PMID- 27484482
OWN - NLM
STAT- MEDLINE
DCOM- 20170613
LR  - 20181113
IS  - 1362-4962 (Electronic)
IS  - 0305-1048 (Print)
IS  - 0305-1048 (Linking)
VI  - 44
IP  - 16
DP  - 2016 Sep 19
TI  - Co-incident insertion enables high efficiency genome engineering in mouse 
      embryonic stem cells.
PG  - 7997-8010
LID - 10.1093/nar/gkw685 [doi]
AB  - CRISPR/Cas9 nucleases have enabled powerful, new genome editing capabilities; 
      however, the preponderance of non-homologous end joining (NHEJ) mediated repair 
      events over homology directed repair (HDR) in most cell types limits the ability 
      to engineer precise changes in mammalian genomes. Here, we increase the 
      efficiency of isolating precise HDR-mediated events in mouse embryonic stem (ES) 
      cells by more than 20-fold through the use of co-incidental insertion (COIN) of 
      independent donor DNA sequences. Analysis of on:off-target frequencies at the 
      Lef1 gene revealed that bi-allelic insertion of a PGK-Neo cassette occurred more 
      frequently than expected. Using various selection cassettes targeting multiple 
      loci, we show that the insertion of a selectable marker at one control site 
      frequently coincided with an insertion at an unlinked, independently targeted 
      site, suggesting enrichment of a sub-population of HDR-proficient cells. When 
      individual cell events were tracked using flow cytometry and fluorescent protein 
      markers, individual cells frequently performed either a homology-dependent 
      insertion event or a homology-independent event, but rarely both types of 
      insertions in a single cell. Thus, when HDR-dependent selection donors are used, 
      COIN enriches for HDR-proficient cells among heterogeneous cell populations. When 
      combined with a self-excising selection cassette, COIN provides highly efficient 
      and scarless genome editing.
CI  - (c) The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic 
      Acids Research.
FAU - Shy, Brian R
AU  - Shy BR
AD  - Department of Biochemistry and Molecular Genetics, University of Illinois at 
      Chicago, Chicago, IL 60607, USA Genome Editing Core, University of Illinois at 
      Chicago, Chicago, IL 60607, USA.
FAU - MacDougall, Matthew S
AU  - MacDougall MS
AD  - Department of Biochemistry and Molecular Genetics, University of Illinois at 
      Chicago, Chicago, IL 60607, USA.
FAU - Clarke, Ryan
AU  - Clarke R
AD  - Department of Biochemistry and Molecular Genetics, University of Illinois at 
      Chicago, Chicago, IL 60607, USA.
FAU - Merrill, Bradley J
AU  - Merrill BJ
AD  - Department of Biochemistry and Molecular Genetics, University of Illinois at 
      Chicago, Chicago, IL 60607, USA Genome Editing Core, University of Illinois at 
      Chicago, Chicago, IL 60607, USA merrillb@uic.edu.
LA  - eng
GR  - R01 HD081534/HD/NICHD NIH HHS/United States
GR  - T32 GM079086/GM/NIGMS NIH HHS/United States
GR  - T32 HL007829/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20160802
PL  - England
TA  - Nucleic Acids Res
JT  - Nucleic acids research
JID - 0411011
RN  - 0 (CRISPR-Associated Proteins)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Animals
MH  - Base Sequence
MH  - CRISPR-Associated Proteins/metabolism
MH  - DNA/genetics
MH  - DNA Breaks, Double-Stranded
MH  - DNA End-Joining Repair/genetics
MH  - Gene Editing
MH  - Genetic Engineering/*methods
MH  - *Genome
MH  - Homologous Recombination/genetics
MH  - Mice
MH  - Mice, Inbred C57BL
MH  - Mouse Embryonic Stem Cells/*metabolism
MH  - Mutagenesis, Insertional/*genetics
MH  - Polymerase Chain Reaction
MH  - Recombinational DNA Repair
PMC - PMC5027516
EDAT- 2016/08/04 06:00
MHDA- 2017/06/14 06:00
PMCR- 2016/08/02
CRDT- 2016/08/04 06:00
PHST- 2016/07/25 00:00 [accepted]
PHST- 2016/02/14 00:00 [received]
PHST- 2016/08/04 06:00 [entrez]
PHST- 2016/08/04 06:00 [pubmed]
PHST- 2017/06/14 06:00 [medline]
PHST- 2016/08/02 00:00 [pmc-release]
AID - gkw685 [pii]
AID - 10.1093/nar/gkw685 [doi]
PST - ppublish
SO  - Nucleic Acids Res. 2016 Sep 19;44(16):7997-8010. doi: 10.1093/nar/gkw685. Epub 
      2016 Aug 2.