When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping.
Alkylglycerol monooxygenase
Ether lipid metabolism
Genomic structural variation
Homologous recombination
Mouse models
Journal
Cell & bioscience
ISSN: 2045-3701
Titre abrégé: Cell Biosci
Pays: England
ID NLM: 101561195
Informations de publication
Date de publication:
16 Mar 2021
16 Mar 2021
Historique:
received:
01
12
2020
accepted:
25
02
2021
entrez:
17
3
2021
pubmed:
18
3
2021
medline:
18
3
2021
Statut:
epublish
Résumé
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. 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. 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.
Sections du résumé
BACKGROUND
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
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
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.
Identifiants
pubmed: 33726865
doi: 10.1186/s13578-021-00566-9
pii: 10.1186/s13578-021-00566-9
pmc: PMC7962373
doi:
Types de publication
Journal Article
Langues
eng
Pagination
54Subventions
Organisme : Austrian Science Fund
ID : P30800
Organisme : Austrian Science Fund
ID : P33333
Organisme : Austrian Science Fund FWF
ID : P 30800
Pays : Austria
Organisme : Austrian Science Fund FWF
ID : P 28769
Pays : Austria
Organisme : Deutsche Forschungsgemeinschaft
ID : CRC1039 A03
Organisme : British Heart Foundation
ID : RG/12/5/29576
Pays : United Kingdom
Organisme : Austrian Science Fund
ID : P28769
Organisme : British Heart Foundation
ID : CH/16/1/32013
Pays : United Kingdom
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