A Combined Human in Silico and CRISPR/Cas9-Mediated in Vivo Zebrafish Based Approach to Provide Phenotypic Data for Supporting Early Target Validation.
A Combined Human in Silico and CRISPR/Cas9-Mediated in Vivo Zebrafish Based Approach to Provide Phenotypic Data for Supporting Early Target Validation.
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DOI：
10.3389/fphar.2022.827686
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发表时间：
2022
影响因子：
5.6
通讯作者：
Bohlooly-Y, Mohammad
中科院分区：
文献类型：
--
作者：
Winter, Matthew J.;Ono, Yosuke;Ball, Jonathan S.;Walentinsson, Anna;Michaelsson, Erik;Tochwin, Anna;Scholpp, Steffen;Tyler, Charles R.;Rees, Steve;Hetheridge, Malcolm J.;Bohlooly-Y, Mohammad
关键词：
心力衰竭的临床异质性挑战了我们对这种疾病潜在遗传机制的理解。在这方面，大规模的患者DNA测序研究已经成为识别潜在遗传因素的宝贵策略。然而，心力衰竭的复杂病因也意味着体内模型对于了解遗传扰动和功能影响之间的联系至关重要，这是验证潜在新药靶点过程的一部分。传统的方法（例如，转基因小鼠）对于评估少量基因是最佳的，但是当识别多个基因时就不太实用了。相比之下，斑马鱼提供了更高通量的体内基因功能评估的巨大潜力，通过对靶标相关性提供更多的信心，促进基因选择，以帮助确定靶标优先级，从而在小鼠中进行明确的功能丧失研究。在这里，我们对人类患者数据使用全外显子组测序和生物信息学来鉴定3个基因（API5， HSPB7和LMO2）与心力衰竭相关，并且预计在疾病病因学中发挥更广泛的作用。然后利用斑马鱼体内CRISPR/ cas9介导的基因突变分析进一步研究了这些基因在心血管系统发育和功能中的作用。我们观察到F0基因敲除的斑马鱼胚胎（crispants）在发生有效体细胞突变后的多重影响，包括心室大小、心包水肿和心室畸形的变化。在lmo2的情况下，也有对心血管功能的显著影响，以及预期的红细胞生成减少。从人体硅和斑马鱼体内评估中获得的数据支持了API5、HSPB7和LMO2在人类心血管疾病中的潜在作用的进一步研究。提出的数据也支持使用人类硅基因变异分析，结合斑马鱼脆化表型，作为评估基因功能的强大方法，作为综合多层次药物靶点验证策略的一部分。
The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts as part of the process for validating potential new drug targets. Traditional approaches (e.g., genetically-modified mice) are optimal for assessing small numbers of genes, but less practical when multiple genes are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation, by providing more confidence in target relevance and facilitating gene selection for definitive loss of function studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including changes in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports further investigation of the potential roles of API5, HSPB7, and LMO2 in human cardiovascular disease. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.
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影响因子：
120.1
作者：
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通讯作者：
Pangalos, Menelas N.
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Gustafson, A. -L.;Stedman, D. B.;Augustine-Rauch, K. A.
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Augustine-Rauch, K. A.
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Augustine-Rauch, Karen A.