| RRC ID |
41306
|
| Author |
Kimura Y, Oda M, Nakatani T, Sekita Y, Monfort A, Wutz A, Mochizuki H, Nakano T.
|
| Title |
CRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells.
|
| Journal |
Sci Rep
|
| Abstract |
Mouse parthenogenetic haploid embryonic stem cells (ESCs) are pluripotent cells generated from chemically activated oocytes. Haploid ESCs provide an opportunity to study the effect of genetic alterations because of their hemizygotic characteristics. However, their further application for the selection of unique phenotypes remains limited since ideal reporters to monitor biological processes such as cell differentiation are missing. Here, we report the application of CRISPR/Cas9-mediated knock-in of a reporter cassette, which does not disrupt endogenous target genes in mouse haploid ESCs. We first validated the system by inserting the P2A-Venus reporter cassette into the housekeeping gene locus. In addition to the conventional strategy using the Cas9 nuclease, we employed the Cas9 nickase and truncated sgRNAs to reduce off-target mutagenesis. These strategies induce targeted insertions with an efficiency that correlated with sgRNA guiding activity. We also engineered the neural marker gene Sox1 locus and verified the precise insertion of the P2A-Venus reporter cassette and its functionality by monitoring neural differentiation. Our data demonstrate the successful application of the CRISPR/Cas9-mediated knock-in system for establishing haploid knock-in ESC lines carrying gene specific reporters. Genetically modified haploid ESCs have potential for applications in forward genetic screening of developmental pathways.
|
| Volume |
5
|
| Pages |
10710
|
| Published |
2015-6-3
|
| DOI |
10.1038/srep10710
|
| PII |
srep10710
|
| PMID |
26039937
|
| PMC |
PMC4454075
|
| MeSH |
Animals
CRISPR-Cas Systems*
Cell Differentiation
Embryonic Stem Cells / cytology
Embryonic Stem Cells / metabolism*
Female
Gene Expression*
Gene Knock-In Techniques
Gene Order
Gene Targeting
Genes, Reporter*
Genetic Loci
Haploidy*
Male
Mice
Mice, Transgenic
RNA, Guide, Kinetoplastida / genetics
SOXB1 Transcription Factors / genetics
|
| IF |
3.998
|
| Times Cited |
20
|
|
WOS Category
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
| Resource |
| DNA material |
CSII-EF-Venus (RDB05552) |
