Journal
PEERJ
Volume 1, Issue -, Pages -Publisher
PEERJ INC
DOI: 10.7717/peerj.230
Keywords
CRISPR/Cas; Haploid; Embryonic stem cells; Genome engineering; Tet1; Tet2; Tet3
Categories
Funding
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- Ministry of Health, Labor and Welfare of Japan
- National Institute of Biomedical Innovation
- Asahi Glass Foundation
- Ichiro Kanehara Foundation
- Takeda Science Foundation
- Grants-in-Aid for Scientific Research [24570153, 24116515, 25650007, 22591816] Funding Source: KAKEN
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Haploid embryonic stem cells (ESCs) are useful for studying mammalian genes because disruption of only one allele can cause loss-of-function phenotypes. Here, we report the use of haploid ESCs and the CRISPR RNA-guided Cas9 nuclease gene-targeting system to manipulate mammalian genes. Co-transfection of haploid ESCs with vectors expressing Cas9 nuclease and single-guide RNAs (sgRNAs) targeting Tet1, Tet2, and Tet3 resulted in the complete disruption of all three genes and caused a loss-of-function phenotype with high efficiency (50%). Co-transfection of cells with vectors expressing Cas9 and sgRNAs targeting two loci on the same chromosome resulted in the creation of a large chromosomal deletion and a large inversion. Thus, the use of the CRISPR systemin combination with haploid ESCs provides a powerful platform to manipulate the mammalian genome.
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