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

Abstract

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.

Genetic models of fibrillinopathies

“…FBN1 variants have been introduced in the pig using genome editing. Our own attempt using CRISPR-Cas9 genome editing to target exon 2 of the pig FBN1 resulted in 2 offspring with an identical homozygous 5-base pair deletion ( Tsang et al 2020 ). The piglets were severely compromised by hydrops fetalis, a complication not reported for MFS or the homozygous mouse models, and likely the result of off-target effects.…”