Markby, G. R. | bioRxiv Science

bioRxiv2020

Severe perinatal hydrops fetalis in genome edited pigs with a biallelic five base pair deletion of the Marfan syndrome gene, FBN1

This paper describes a genome editing project using CRISPR-Cas9. The objective was to create a large animal model of human Marfan syndrome by targeting the FBN1 gene of the pig, Sus scrofa, using a single guide and non-homologous end joining which was expected to create short insertion or deletion mutations at the 5 end of the gene. The editing successfully created a five base pair deletion in exon 2 of FBN1, which was homozygous in two animals. However, the phenotype of these piglets was unexpected, since they showed none of the signs consistent with Marfan syndrome but both suffered extreme hydrops fetalis with a large amount of fluid located under the skin and in the abdomen. One of the edited piglets was stillborn and the other was euthanised at birth on welfare grounds. It is likely that this result was due to unanticipated on- or off-target mutations, possibly in the GLDN gene 3 megabases away from FBN1. This result provides more evidence for unexpected outcomes of CRISPR- Cas9 gene editing and supports the proposal that all genome edited individuals should be subjected to strategies to track the CRISPR footprint, such as whole genome sequencing, before being used for further experimentation or in clinical applications.

molecular biology↗

bioRxiv2020

Gene expression in the cardiovascular system of the domestic sheep (Ovis aries); a new tool to advance our understanding of cardiovascular disease.

Large animal models are of increasing importance in cardiovascular disease research as they demonstrate more similar cardiovascular features (in terms of anatomy, physiology and size) to humans than do rodent species. The maintenance of a healthy cardiovascular system requires expression of genes that contribute to essential biological activities and repression of those that are associated with functions likely to be detrimental to cardiovascular homeostasis. In this study we have used the transcriptome of the sheep, which has been utilised extensively to model human physiology and disease, to explore genes implicated in the process of vascular calcification. Vascular calcification is a major disruption to cardiovascular homeostasis where tissues of the cardiovascular system undergo ectopic calcification and consequent dysfunction. We investigate the gene expression profiles of genes involved in vascular calcification in a wide array of cardiovascular tissues and across multiple developmental stages, using RT-qPCR. The majority of transcriptomic studies on the mammalian cardiovascular system to date have focused on regional expression of specific genes. Here we also use RNA sequencing results from the sheep heart and cardiac valves to further explore the transcriptome of the cardiovascular system in this large animal. Our results demonstrate that there is a balance between genes that promote and those that suppress mineralisation during development and across cardiovascular tissues. We show extensive expression of genes encoding proteins involved in formation and maintenance of the extracellular matrix in cardiovascular tissues, and high expression of haematopoietic genes in the cardiac valves. Our analysis will support future research into the functions of implicated genes in the development of vascular calcification, and increase the utility of the sheep as a large animal model for understanding cardiovascular disease. This study provides a foundation to explore the transcriptome of the developing cardiovascular system and is a valuable resource for the fields of mammalian genomics and cardiovascular research.

genomics↗