When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping

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.

CRISPR-LRS for mapping transgenes in the mouse genome

“…Of the available strategies to map transgenes, WGS (2, 9) and TLA (1, 5) have the most traction. However with WGS, most of the sequence data is uninformative and sequence depth is substantial with ~2-5x coverage of the host genome (2,9). Here, with only ~1.8Gb of cumulative sequence data, CRISPR-LRS mapped four mouse lines with two lines yielding PCR validated chromosome coordinates.…”

“…Inverse PCR has high failure rates due to concatemerization of most transgenes (8). Recently, whole genome sequencing (WGS) with long-read sequencing platforms, PacBio or Oxford Nanopore Technologies (ONT), have been used to map transgenes (2,9). However, even with ~7.5Gb of sequencing data for ~3x coverage of the mouse genome, researchers cannot be assured to find reads that identify the breakpoint of the transgene.…”

AGMO Inhibitor Reduces 3T3-L1 Adipogenesis

“…AGMO inhibition might reduce the expansion of visceral fat, if such antiproliferative effects were to occur in vivo. In a previous study, lipid droplets were absent in ether-lipid-deficient mice and were restored with an AKG-rich diet that also alleviated the progression of the pathology of these mice in testis and adipose tissue [ 62 ], both sites with high AGMO expression [ 63 ].…”

Concatenation of Transgenic DNA: Random or Orchestrated?

“…In this review, we will not focus on it, because TLA mostly provides information about the integration site and the surrounding genomic locus. Undoubtedly, this method will be important for systematic analysis of the thousands of archived transgenic mouse lines [ 52 ] to chart integration site preference and analyze collateral genomic damage, including the pervasive presence of the flanking chromosomal duplications that could be found in random and targeted integration approaches [ 80 , 129 , 130 , 139 , 140 ].…”