Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development

• DOI: 10.1371/journal.pbio.3000350
• Publication Date: 2019-07-02T17:23:19Z
• Authors: Meizhu Bai; Yujiao Han; Yuxuan Wu; Jiaoyang Liao; Lin Li; Lijun Wang; Qing Li; Wenhui Xing; Luonan Chen; Weiguo Zou; Jinsong Li
• Keywords: Homeodomain Proteins; Mice, Knockout; 0301 basic medicine; Bone Development; QH301-705.5; Relaxin; Methods and Resources; Gene Expression Regulation, Developmental; Mouse Embryonic Stem Cells; Haploidy; Hematopoietic Cell Growth Factors; Mice; 03 medical and health sciences; Gene Targeting; Animals; Lectins, C-Type; Genetic Testing; Biology (General); CRISPR-Cas Systems; Cells, Cultured; Transcription Factors

Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only lower organisms. Here, we develop a targeted genetic approach mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced mutant semi-cloned (SC) pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 an single guide RNA (sgRNA) library targeting 72 preselected one step screened bone-development–related skeletal analysis at birth. This yielded 4 genes: Zic1 Clec11a, which required bone development, Rln1 Irx5, had not been previously considered. Whereas Rln1−/− exhibited small size birth, Irx5−/− showed abnormalities both postnatal adult phases due to decreased mass increased marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis inhibits adipogenesis suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic opens new avenues interrogation processes mice.