Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
The Musculoskeletal Genetics Laboratory, The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel.
J Bone Miner Res. 2021 Sep;36(9):1749-1764. doi: 10.1002/jbmr.4327. Epub 2021 May 19.
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. © 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).
全基因组关联研究（GWAS）提高了我们对骨质疏松等常见复杂疾病遗传结构的理解。然而，为了将候选基因的功能性骨骼贡献归因于与骨质疏松相关的特征，需要进行高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，传统上在稳定的生殖系敲除（KO）突变体中进行表型分析。最近，第一代（F0）嵌合体突变斑马鱼（所谓的crispants）重现了生殖系 KO 的表型。为了证明通过crispant 筛选对骨质疏松候选基因进行功能验证的可行性，我们比较了crispant 和稳定 KO 斑马鱼模型的 lrp5 基因。在人类中，Wnt 信号通路的共受体 LRP5 的隐性失功能突变导致骨质疏松-假瘤综合征。此外，几项 GWAS 研究发现 LRP5 是与骨质疏松相关表型的主要风险位点。在这项研究中，我们表明早期 lrp5 KO 幼虫显示出脊索矿化减少和头部软骨畸形。成年骨骼的定量微计算机断层扫描（micro-CT）扫描和质谱元素分析显示椎骨体积和骨矿化减少，这是骨质疏松的标志性特征。此外，再生鳍组织显示 lrp5 KO 成鱼中 Wnt 信号活性降低。我们接下来比较了 lrp5 突变体和 crispants。成年 crispant 组织的下一代测序分析显示平均移框突变率为 76%，导致 Lrp5 蛋白水平显著降低。这些 crispants 通常表现出更温和但仍然高度可比的骨骼表型，以及与 lrp5 KO 突变体相比，Wnt 途径反应降低。总之，我们通过对 LRP5 相关原发性骨质疏松症的忠实建模表明，crispant 筛选在斑马鱼中是快速筛选骨质疏松候选基因的有前途的方法。 2021 年作者。骨与矿物研究杂志由 Wiley 期刊出版公司代表美国骨与矿物研究协会（ASBMR）出版。
