Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.Grant-in-Aid for Scientific Research(B), Japan Society for the Promotion of Science (JSPS KAKENHI 17H03568) Fumihiro Sugiyama Grant-in-Aid for Scientific Research(S), Japan Society for the Promotion of Science (JSPS KAKENHI 26221004) Satoru Takahashi Grant-in-Aid for Scientific Research(C), Japan Society for the Promotion of Science (JSPS KAKENHI 17K07130) Hiroyoshi Iseki Grant-in-Aid for Young Scientists (B), Japan Society for the Promotion of Science (JSPS KAKENHI 19K16020) Tra Thi Huong Dinh Grant-in-Aid for Scientific Research(A), Japan Society for the Promotion of Science (JSPS KAKENHI 20H00444) Fumihiro Sugiyama The Cooperative Research Project Program of Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA Center), University of Tsukuba, Japan (182107) Fumihiro Sugiyam

Supplementary Table S1.;Supplementary Table S2. from Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions

Sequence of sgRNAs used in this study.;Sequence of primers used in this study

Generation of bicistronic reporter knockin mice for visualizing germ layers

Knockout mouse models are commonly used in developmental biology to investigate the functions of specific genes, and the knowledge obtained in such models has yielded insights into the molecular mechanisms underlying developmental processes. Gastrulation is the most dynamic process in embryogenesis during which differentiation into three germ layers occurs. However, the functions of genes involved in gastrulation are not completely understood. One major reason for this is the technical difficulty of embryo analysis to understand germ layer location. We have generated three reporter mouse strains in which the germ layers are distinguished by different fluorescent reporters. Using CRISPR/Cas9 genome editing in mouse zygotes, the fluorescent reporter genes, EGFP, tdTomato, and TagBFP including 2A peptide sequences were knocked into the appropriate sites before the stop codon of the Sox17 (endoderm marker), Otx2 (ectoderm marker), and T (mesoderm marker) genes, respectively. Founder mice were successfully generated in the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter strains. Further, homozygous knockin mice of all strains appeared morphologically normal and were fertile. On stereomicroscopic analysis, fluorescent signals were detected in a germ layer-specific manner from heterozygous embryos at embryonic day (E) 6.5–8.5 in all strains, and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation