Abstract
Studying the early function of essential genes is an important and challenging problem in developmental biology. Here, we established a method for rapidly inducing CRISPR-Cas9-mediated mutations in one blastomere of two-cell stage embryos, termed 2-cell embryo-CRISPR-Cas9 injection (2CC), to study the in vivo function of essential (or unknown) genes in founder chimeric mice. By injecting both Cre mRNA and CRISPR-Cas9 targeting the gene of interest into fluorescent reporter mice, the 2CC method can trace both wild-type and mutant cells at different developmental stages, offering internal control for phenotypic analyses of mutant cells. Using this method, we identified novel functions of the essential gene Tet3 in regulating excitatory and inhibitory synaptic transmission in the developing mouse cerebral cortex. By generating chimeric mutant mice, the 2CC method allows for the rapid screening of gene function in multiple tissues and cell types in founder chimeric mice, significantly expanding the current armamentarium of genetic tools.
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Acknowledgements
We thank Yi Zeng (Shanghai Institute of Biochemistry and Cell Biology) for kindly providing mT/mG mice. This study was supported by the Ministry of Science and Technology of China (2014CB964803 and 2015AA020307 to JL), the National Natural Science Foundation of China (31530048 and 81672117 to JL, 31530030 to XY and 31601163 to KW), the Chinese Academy of Sciences (XDB19010204 to JL and XDB02010000 to XY), Shanghai Municipal Commission for Science and Technology (16JC1420500 to JL) and China Postdoctoral Science Foundation (2016M601661 to KW).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Table S1
In vitro and in vivo Development of Two-cell Embryos after Injection of Cre mRNA into One Blastomere*. (PDF 107 kb)
Supplementary information, Table S2
CRISPR-Cas9-Mediated Tet1 or Tet3 Targeting in One Blastomere of Two-cell Embryos*. (PDF 115 kb)
Supplementary information, Table S3
Multiple sgRNAs Mediated Tet3, Tyr or Gata4 Targeting in One Blastomere of Two-cell Stage Embryos*. (PDF 120 kb)
Supplementary information, Table S4
Off-target Analysis in Chimeric Mice Carrying Tet3 Mutation Generated Using Multiple sgRNAs. (PDF 133 kb)
Supplementary information, Figure S1
Chimerism Analysis of Embryos and Mice Derived after Cre mRNA Injection into One Blastomere of Two-cell Stage Embryos. (PDF 3662 kb)
Supplementary information, Figure S2
Analysis of Tet1 or Tet3 Targeted mT+/mG+ ESC Lines and Mice. (PDF 2411 kb)
Supplementary information, Figure S3
Multiple sgRNAs Mediated Tyr or Gata4 Targeting. (PDF 5130 kb)
Supplementary information, Figure S4
CRISPR-Cas9-mediated Tet3 Targeting Using the Ai9 Reporter Line. (PDF 1141 kb)
Supplementary information, Figure S5
Whole-cell Recordings from the Somatosensory Cortex of Chimeric Ai9 mice. (PDF 387 kb)
Supplementary information, Figure S6
Gene Expression And Local Methylation Analysis of Candidate Target Genes in Tet3 Mutant Chimeric Mice. (PDF 884 kb)
Supplementary information, Data S1
Supplemental experimental procedures (PDF 133 kb)
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Wang, L., Li, MY., Qu, C. et al. CRISPR-Cas9-mediated genome editing in one blastomere of two-cell embryos reveals a novel Tet3 function in regulating neocortical development. Cell Res 27, 815–829 (2017). https://doi.org/10.1038/cr.2017.58
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DOI: https://doi.org/10.1038/cr.2017.58
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