Lrp5 Mutant and Crispant Zebrafish Faithfully Model Human Osteoporosis, Establishing the Zebrafish as a Platform for CRISPR-Based Functional Screening of Osteoporosis Candidate Genes
Lrp5 Mutant and Crispant Zebrafish Faithfully Model Human Osteoporosis, Establishing the Zebrafish as a Platform for CRISPR-Based Functional Screening of Osteoporosis Candidate Genes
复制标题
Lrp5 突变体和 Crispant 斑马鱼忠实地模拟人类骨质疏松症，将斑马鱼建立为基于 CRISPR 的骨质疏松症候选基因功能筛选平台
DOI：
10.1002/jbmr.4327
复制
发表时间：
2021-05-19
影响因子：
6.2
通讯作者：
Coucke, Paul J.
中科院分区：
文献类型：
--
作者：
Bek, Jan Willem;Shochat, Chen;Coucke, Paul J.
全基因组关联研究（GWAS）提高了我们对常见复杂疾病（如骨质疏松症）的遗传结构的理解。然而，要将候选基因的功能性骨骼贡献归因于阿尔茨海默病相关性状，需要进行有效且具有成本效益的体内功能测试。这可以通过基于CRISPR的反向遗传筛选来实现，其中表型分析传统上在稳定的种系敲除（KO）突变体中进行。最近的研究表明，第一代（F0）嵌合突变体斑马鱼（所谓的crispants）重现了种系科斯的表型。为了证明通过crispant筛选对骨质疏松症候选基因进行功能验证的可行性，我们比较了crispant与稳定的Lrp 5基因KO斑马鱼模型。在人类中，LRP 5（Wnt信号通路中的共受体）的隐性功能丧失突变导致神经胶质瘤-假神经胶质瘤综合征。此外，几项GWAS研究将LRP 5确定为糖尿病相关表型的主要风险位点。在这项研究中，我们发现，早期阶段lrp 5 KO幼虫显示脊索矿化减少和头部软骨畸形。对成人骨骼的定量微计算机断层扫描（micro-CT）和质谱元素分析显示椎体骨体积和骨矿化减少，这是骨质疏松症的标志性特征。此外，再生的鳍组织在lrp 5 KO成体中显示降低的Wnt信号传导活性。我们接下来比较了lrp 5突变体与卷曲体。对成年松脆组织的下一代测序分析显示，平均框外突变率为76%，导致Lrp 5蛋白水平大幅降低。与lrp 5 KO突变体相比，这些卷曲体通常显示出更温和但仍然高度可比的骨骼表型和类似降低的Wnt途径应答。总之，我们通过对LRP 5相关原发性骨质疏松症的忠实建模表明，在斑马鱼中进行crispant筛选是一种很有前途的方法，可用于骨质疏松症候选基因的快速功能筛选。(C)2021年美国骨与矿物质研究学会。(C)2021作者《骨与矿物质研究杂志》由Wiley Periodicals LLC代表美国骨与矿物质研究学会（ASBMR）出版。
Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. (C) 2021 American Society for Bone and Mineral Research. (C) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).