When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping

• DOI: 10.1186/s13578-021-00566-9
• Publication Date: 2021-03-16T12:11:47Z
• Authors: Sabrina Sailer; Stefan Coassin; Katharina Lackner; Caroline Fischer; Eileen McNeill; Gertraud Streiter; Christian Kremser; Manuel Maglione; Catherine M. Green; Daniela Moralli; Alexander R. Moschen; Markus A. Keller; Georg Golderer; Gabriele Werner-F...; Irmgard Tegeder; Keith M. Channon; Benjamin Davies; Ernst R. Werner; Katrin Watschinger
• Keywords: Segmental duplication; Structural Variation

Genome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts gene segment duplications) that lead mutations not only close proximity the site but also outside. Only genomes of few engineered mouse strains have sequenced. Since role ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) physiology and pathophysiology remains enigmatic, we created a knockout model for AGMO EUCOMM stem cells unforeseen genotyping issues did agree with Mendelian distribution activity data prompted an in-depth genomic validation model.We report tandem duplication event occurred during generation Agmo knockout-first allele by recombination. low homology was seen between breakpoints. While single copy recombinant 18 kb cassette integrated correctly around exon 2 gene, whole genome nanopore sequencing revealed 94 locus contains wild-type exons 1-3. The fooled routine PCR, could be resolved qPCR-based genotyping, targeted amplification sequencing. Despite this event, lacks can therefore used study its physiological role.A at exact detected conventional quality control filters such as FISH long-range PCR over sites. Nanopore provides cost convenient method detect underrated off-target effects, suggesting use additional assessment other organisms.