When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping
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Le résumé fourni par la source
BACKGROUND: 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 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. RESULTS: We 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. CONCLUSIONS: A 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.
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Le contrôle bibliographique ouvert
DOI retrouvé dans Crossref DOI retrouvé ; titre concordant.
- Titre Crossref
- When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping
- Date Crossref
- 16/03/2021
- Éditeur
- Springer Science and Business Media LLC
- Type
- journal-article
Ce recoupement confirme des métadonnées liées au DOI. Il ne confirme ni la méthode ni les conclusions de l’étude, et il ne compte pas comme une seconde source scientifique indépendante.
Où se fait cette recherche
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Innsbruck Medical University pays non établi dans la noticeUniversité ou école supérieure
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Universität Innsbruck pays non établi dans la noticeUniversité ou école supérieure
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Goethe University Frankfurt pays non établi dans la noticeUniversité ou école supérieure
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Centre for Human Genetics pays non établi dans la noticeStructure de recherche
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British Heart Foundation pays non établi dans la noticeOrganisation à but non lucratif
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University of Oxford British Heart Foundation Centre of Research Excellence pays non établi dans la noticeUniversité ou école supérieure
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Medical University of Innsbruck Institute of Biological Chemistry pays non établi dans la noticeUniversité ou école supérieure
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Institute of Clinical Pharmacology of the Medical Faculty pays non établi dans la noticeUniversité ou école supérieure
Innsbruck Medical University, Universität Innsbruck et Goethe University Frankfurt, avec 5 autres affiliations.
Une affiliation ne permet pas de déduire la nationalité d’un auteur.