A Combined Human in Silico and CRISPR/Cas9-Mediated in Vivo Zebrafish Based Approach to Provide Phenotypic Data for Supporting Early Target Validation

Abstract

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.

The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

“…During blastula stages, Flot1b/Reg2b and Flot2a/Reg1a are predominantly expressed in the zebrafish ( Von Philipsborn et al, 2005 ). Therefore, we used our CRISPR/Cas9- KO strategy in zebrafish embryos ( Winter et al, 2022 ) to generate F0 knockout zebrafish embryos (Crispants) for Flot1b/2 a. ( Figure 6G ).…”

“…Prior to injection, gene-specific crRNA and tracrRNA (Integrated DNA Technologies Inc Coralville, United States) were diluted to a final concentration of 12 μM in nuclease-free duplex buffer and the resultant gRNA mixture incubated at 95°C for 5 min ( Winter et al, 2022 ). Immediately prior to use, 5 μl of the gRNA mixture was mixed with Cas9-NLS protein (final concentration of 5 μM.…”

Beyond compliance: harmonising research and husbandry practices to improve experimental reproducibility using fish models

“…Further advantages include the availability of a sequenced and well‐annotated genome and the ability to manipulate gene function readily and to generate transgenic lines through techniques such as the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9) system (Schartl, 2014; Zhu & Ge, 2018). Application of CRISPR/Cas9 gene manipulation in the zebrafish includes early drug target validation for human disease treatment (Winter et al ., 2022).…”

“…There are also 3Rs (replacement, reduction and refinement) benefits for adopting fish versus rodents as experimental models. This includes that fish early life stages do not constitute a regulated procedure, and skin/body translucency in many fish early life stages allows for real‐time non‐invasive visualisation of development (Kimmel et al ., 1995) and chemical effect and disease process (Yan et al ., 2019; Winter et al ., 2022). For the zebrafish, non‐protected life stages (i.e.…”

“…Today, zebrafish models are used by scientists in a wide range of disciplines including embryology (Pathak & Barresi, 2020), physiology (Kwong, Kumai & Perry, 2014), neuroscience (Hall et al ., 2014), behaviour (McArthur, Chow & Fetcho, 2020), epigenetics (Cavalieri & Spinelli, 2017), ecotoxicology (Bambino & Chu, 2017), disease biology (Bailone et al ., 2020) and the effects of climate change (Pinheiro et al ., 2021). The ability to create genetically selected and/or manipulated zebrafish lines has furthermore enabled significant advances in precision for ecotoxicology (Cassar et al ., 2020), disease processes (Adamson, Sheridan & Grierson, 2018) and human drug validation studies (Winter et al ., 2022).…”