State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.
State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.
PLoS Biol. 2019 Jul 2;17(7):e3000350. doi: 10.1371/journal.pbio.3000350. eCollection 2019 Jul.
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 clustered 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 one step and screened for bone-development-related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a, which are required for bone development, and Rln1 and Irx5, which had not been previously considered. Whereas Rln1-/- 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.
诱变筛选在鉴定发育过程中涉及的关键基因方面非常有效。然而，这种筛选仅在低等生物中成功。在这里，我们通过结合雄激素单倍体胚胎干细胞（AG-haESCs）和成簇规律间隔短回文重复/CRISPR 相关蛋白 9（CRISPR-Cas9）技术，在小鼠中开发了一种靶向遗传筛选方法。我们通过卵母细胞注射携带组成型表达 Cas9 和靶向 72 个预选基因的单指导 RNA（sgRNA）文库的 AG-haESCs，一步产生突变半克隆（SC）小鼠池，并通过出生时的骨骼分析筛选与骨骼发育相关的基因。这产生了 4 个基因：Zic1 和 Clec11a，它们是骨骼发育所必需的，而 Rln1 和 Irx5 以前没有被认为是必需的。虽然 Rln1-/-小鼠仅在出生时表现出骨骼较小，但 Irx5-/-小鼠由于骨量减少和骨髓脂肪生成增加，在出生后和成年期都表现出骨骼异常。从机制上讲，同源异形盒基因 5（IRX5）通过抑制过氧化物酶体增殖物激活受体 γ（PPARγ）激活来促进成骨细胞生成并抑制脂肪生成。因此，AG-haESC 介导的功能诱变筛选为小鼠发育过程中的遗传研究开辟了新途径。
