Abstract

Genetically engineered mouse models are invaluable tools to investigate gene and genome function and elucidate disease mechanisms. The availability of new programmable, targeted nucleases has vastly increased the accessibility of genome engineering.

Across the scale, from small, subtle and seamless modifications to large complex multifunctional alleles, nuclease mediated engineering introduces novel challenges for genome validation. We must not only identify and scrutinise the targeted allele, but also exclude unexpected mutations and off-target effects, all within the complex genetic architecture of mosaic founders and the heterogeneous lineages of their offspring.

Our group has developed validation strategies, combining a variety of methodologies to confirm precision engineering at the targeted locus and, importantly, screen out animals with undesired mutations including off-target editing, random integrations, chromosomal rearrangements and large deletions. We will describe how we identify these mutations and validate correctly engineered alleles by applying methodologies including Sanger sequencing, long-read Nanopore sequencing and digital droplet PCR along with traditional PCR techniques.