Journal
BIOLOGY OF REPRODUCTION
Volume 104, Issue 1, Pages 223-233Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/biolre/ioaa176
Keywords
chimeric mouse; germ cell; spermatozoon; CRISPR/Cas9; Nanos3; embryonic stem cell; Dnmt3b; blastocyst complementation
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Funding
- KAKENHI [19K16017, JP16H06146, JP18H04891, JP25112009, JP19H05758]
- Epigenome Manipulation Project of the All-RIKEN Projects
- Grants-in-Aid for Scientific Research [19K16017] Funding Source: KAKEN
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A CRISPR/Cas9 system utilizing three guide RNAs targeting Nanos3 was able to generate chimeric mice with spermatozoa fully derived from ESCs, confirming that Dnmt3b is not essential for male germ cell development.
Conditional knockout (cKO) mice have contributed greatly to understanding the tissue- or stage-specific functions of genes in vivo. However, the current cKO method requires considerable time and effort because of the need to generate two gene-modified mouse strains (Cre transgenic and loxP knockin) for crossing. Here, we examined whether we could analyze the germ cell-related functions of embryonic lethal genes in F0 chimeric mice by restricting the origin of germ cells to mutant embryonic stem cells (ESCs). We confirmed that the full ESC origin of spermatozoa in fertile chimeric mice was achieved by the CRISPR/Cas9 system using three guide RNAs targeting Nanos3, which induced germ cell depletion in the host blastocyst-derived tissues. Among these fertile chimeric mice, those from male ESCs with a Dnmt3b mutation, which normally causes embryo death, also produced F1 mice derived exclusively from the mutant ESCs. Thus, our new chimeric strategy readily revealed that Dnmt3b is dispensable for male germ cell development, in agreement with a previous cKO study. Our new approach enables us to analyze the germ cell functions of embryonic lethal genes in the F0 generation without using the current cKO method. Summary Sentence A CRISPR/Cas9 system using three guide RNAs targeting Nanos3 enabled the production of chimeric mice with spermatozoa fully derived from ESCs and confirmed that Dnmt3b is not essential for male germ cell development. [GRAPHICS]
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