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

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（mutagenic screening）是鉴定发育过程相关关键基因的有效手段，但此类筛选仅在低等生物中得以成功应用。本研究开发了一种针对小鼠的靶向遗传筛选方法，通过结合孤雄单倍体胚胎干细胞（androgenetic haploid embryonic stem cells，AG-haESCs）与成簇规律间隔短回文重复序列/CRISPR相关蛋白9（clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9，CRISPR-Cas9）技术实现。我们通过一步法向卵母细胞注射携带组成型表达Cas9与靶向72个预选基因的单向导RNA（single guide RNA，sgRNA）文库的AG-haESCs，构建了突变型半克隆（SC）小鼠库，并通过出生时的骨骼分析筛选骨发育相关基因。最终获得4个候选基因：Zic1与Clec11a为骨发育必需基因，Rln1与Irx5则此前未被报道与骨发育相关。其中，Rln1基因敲除（Rln1−/−）小鼠仅在出生时表现出骨骼尺寸偏小的表型，而Irx5基因敲除（Irx5−/−）小鼠因骨量减少与骨髓脂肪生成增加，在出生后及成年阶段均出现骨骼异常。机制研究表明，艾洛魁同源盒5（iroquois homeobox 5，IRX5）通过抑制过氧化物酶体增殖物激活受体γ（peroxisome proliferator activated receptor γ，PPARγ）的激活，促进成骨细胞生成并抑制脂肪生成。综上，基于AG-haESCs的功能性诱变筛选为小鼠发育过程的遗传解析开辟了新途径。