Sabrina Sailer1, Stefan Coassin2, Ernst R Werner1, Katrin Watschinger3,4, Katharina Lackner1, Caroline Fischer5, Eileen McNeill6,7, Gertraud Streiter2, Christian Kremser8, Manuel Maglione9, Catherine M Green6, Daniela Moralli6, Alexander R Moschen10, Markus A Keller11, Georg Golderer1, Gabriele Werner-Felmayer1, Irmgard Tegeder5, Keith M Channon6,7, Benjamin Davies6. 1. Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria. 2. Institute of Genetic Epidemiology, Department of Genetics and Pharmacology, Medical University of Innsbruck, Innsbruck, Austria. 3. Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria. katrin.watschinger@i-med.ac.at. 4. Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria. katrin.watschinger@i-med.ac.at. 5. Institute of Clinical Pharmacology of the Medical Faculty, Goethe-University, Frankfurt (Main), Germany. 6. Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom. 7. Division of Cardiovascular Medicine, British Heart Foundation Centre of Research Excellence, University of Oxford, Oxford, United Kingdom. 8. Department of Radiology, Medical University of Innsbruck, Innsbruck, Austria. 9. Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria. 10. Department of Internal Medicine I, Gastroenterology, Endocrinology and Metabolism, Medical University of Innsbruck, Innsbruck, Austria. 11. Institute of Human Genetics, Medical University of Innsbruck, Innsbruck, Austria.
Abstract
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.
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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