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

by Meizhu Bai, Yujiao Han, Yuxuan Wu, Jiaoyang Liao, Lin Li, Lijun Wang, Qing Li, Wenhui Xing, Luonan Chen, Weiguo Zou, Jinsong Li Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and cl ustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in on e step and screened for bone-development–related genes through skeletal analysis at birth. This yielded 4 genes:Zic1 andClec11a, which are required for bone development, andRln1 andIrx5, which had not been previously considered. WhereasRln1−/− mice exhibited small skeletal size only at birth,Irx5−/− mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice.
Source: PLoS Biology: Archived Table of Contents - Category: Biology Authors: Source Type: research