Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development
Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development
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通过单倍体胚胎干细胞对小鼠进行靶向遗传筛查，鉴定骨发育中的关键基因
DOI：
10.1371/journal.pbio.3000350
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发表时间：
2019-07-01
期刊：
影响因子：
9.8
通讯作者：
Li, Jinsong
中科院分区：
文献类型：
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作者：
Bai, Meizhu;Han, Yujiao;Li, Jinsong
诱变筛选对于鉴定参与发育过程的关键基因是有力的。然而，这样的筛选仅在低等生物中是成功的。在这里，我们通过结合雄激素单倍体胚胎干细胞（AG-haESCs）和成簇规则间隔回文重复序列/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的Cas9的AG-haESC和一步靶向72个预选基因的单向导RNA（sgRNA）文库来产生突变半克隆（SC）小鼠池，并通过出生时的骨骼分析筛选骨发育相关基因。这产生了4个基因：骨骼发育所需的Zic 1和Clec 11 a，以及之前未考虑过的Rln 1和Irx 5。Rln 1 −/−小鼠仅在出生时表现出较小的骨骼尺寸，而Irx 5 −/−小鼠由于骨量减少和骨髓脂肪生成增加而在出生后和成年阶段均表现出骨骼异常。在机制上，易洛魁同源框5（IRX 5）通过抑制过氧化物酶体增殖物激活受体γ（PPARγ）活化来促进成骨细胞生成并抑制脂肪生成。因此，AG-haESC介导的功能性诱变筛选为小鼠发育过程的遗传询问开辟了新的途径。
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.