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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.

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

Alkylglycerol monooxygenase (AGMO; EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl ether bond of alkylglycerols in humans. It is an integral membrane protein with nine predicted transmembrane domains. We attempted to express and purify full-length and truncated forms of AGMO in Escherichia coli. Full-length AGMO could not be expressed in three different E. coli expression strains, three different expression vectors and several induction systems. We succeeded, however, in expression of three N-terminally strep-tagged truncated forms, named active sites 1, 2 and 3, with 205, 134 and 61 amino acids, respectively. Active site 1 fragment, containing two predicted transmembrane regions, a membrane associated region and all known amino acid residues important for catalytic activity, was not fully soluble even in 8 M urea. Active site 2 containing only one predicted membrane associated domain required 8 M urea for solubilisation and eluted in gel filtration in 1 M urea as a trimer. Active site 3 with no hydrophobic domain eluted in gel filtration in 1 M urea as monomer and dimer. These results show that even truncated forms of AGMO are barely soluble when expressed in E. coli and show a high tendency for aggregation.

Current knowledge distinguishes three classes of tetrahydrobiopterin-dependent enzymes as based on protein sequence similarity. These three protein sequence clusters hydroxylate three types of substrate atoms and use three different forms of iron for catalysis. The first class to be discovered was the aromatic amino acid hydroxylases, which, in mammals, include phenylalanine hydroxylase, tyrosine hydroxylase, and two isoforms of tryptophan hydroxylases. The protein sequences of these tetrahydrobiopterin-dependent aromatic amino acid hydroxylases are significantly similar, and all mammalian aromatic amino acid hydroxylases require a non-heme-bound iron atom in the active site of the enzyme for catalysis. The second classes of tetrahydrobiopterin-dependent enzymes to be characterized were the nitric oxide synthases, which in mammals occur as three isoforms. Nitric oxide synthase protein sequences form a separate cluster of homologous sequences with no similarity to aromatic amino acid hydroxylase protein sequences. In contrast to aromatic amino acid hydroxylases, nitric oxide synthases require a heme-bound iron for catalysis. The alkylglycerol monooxygenase protein sequence was the most recent to be characterized. This sequence shares no similarity with aromatic amino acid hydroxylases and nitric oxide synthases. Motifs contained in the alkylglycerol monooxygenase protein sequence suggest that this enzyme may use a di-iron center for catalysis.

The tetrahydrobiopterin-dependent degradation of ether lipids by alkylglycerol monooxygenase (AGMO) produces fatty aldehydes, which are toxic to cells. Therefore, it is of great physiological importance that these harmful compounds are converted into their corresponding, less toxic fatty acids by fatty aldehyde dehydrogenase (FALDH). Dysfunction of this enzyme causes Sjögren-Larsson syndrome. This severe inherited disorder is accompanied by symptoms such as ichthyosis, mental retardation and spasticity. Surprisingly, fatty alcohols and not fatty aldehydes were found to accumulate in fibroblasts of Sjögren-Larsson syndrome patients, suggesting that there can be wide-ranging alterations in the lipid composition of patient cells. In particular, this has to be considered when searching for possible treatment options for patients suffering from Sjögren-Larsson syndrome. For example, inhibition of fatty aldehyde producing ether lipid degradation would have multiple implications on ether lipid- and fatty alcohol-mediated signalling pathways.