Abstract
BackgroundGenome 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 or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model.ResultsWe report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1–3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role.ConclusionsA duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.
Highlights
Genome editing in mice using either classical approaches like homologous recombination or CRISPR/ Cas9 has been reported to harbor off target effects that lead to mutations in close proximity to the target site and outside
Genetic characterization of 40 transgenic mouse lines demonstrated that large deletions and structural variations frequently occurring at the integration sites might interfere with the obtained results and would require careful selection of controls [10]
Generation and validation of Agmo‐deficient mice We generated Agmo knockout-first mice (Agmo-lacZ) using EUCOMM stem cells harboring a transgenic cassette with conditional potential (Fig. 1a)
Summary
Genome editing in mice using either classical approaches like homologous recombination or CRISPR/ Cas has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations in close proximity to the target site and outside. The first transgenic mice were created in 1980 by DNA microinjections into fertilized eggs [2] This method was soon replaced by another approach using gene targeting by homologous recombination in mouse embryonic stem cells [3]. After this milestone, the Cre/loxP system was added for somatic mutations to study tissue specific effects [4]. Genetic characterization of 40 transgenic mouse lines demonstrated that large deletions and structural variations frequently occurring at the integration sites might interfere with the obtained results and would require careful selection of controls [10]
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