Sailer Sabrina, Coassin Stefan, Lackner Katharina, Fischer Caroline, McNeill Eileen, Streiter Gertraud, Kremser Christian, Maglione Manuel, Green Catherine M, Moralli Daniela, Moschen Alexander R, Keller Markus A, Golderer Georg, Werner-Felmayer Gabriele, Tegeder Irmgard, Channon Keith M, Davies Benjamin, Werner Ernst R, Watschinger Katrin
Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.
Institute of Genetic Epidemiology, Department of Genetics and Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.
Cell Biosci. 2021 Mar 16;11(1):54. doi: 10.1186/s13578-021-00566-9.
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.
据报道，使用同源重组或CRISPR/Cas9等经典方法在小鼠中进行基因组编辑存在脱靶效应（插入/缺失、移码或基因片段重复），这不仅会导致在靶位点附近产生突变，还会在靶位点以外产生突变。只有少数工程小鼠品系的基因组进行了测序。由于醚脂裂解酶烷基甘油单加氧酶（AGMO）在生理和病理生理中的作用仍不清楚，我们使用欧洲条件性小鼠突变计划（EUCOMM）干细胞创建了AGMO基因敲除小鼠模型，但意外的基因分型问题（不符合孟德尔遗传分布和酶活性数据）促使我们对该小鼠模型进行深入的基因组验证。
我们报告了在通过同源重组产生Agmo基因敲除首个等位基因的过程中发生的基因片段串联重复事件。断点之间仅观察到低同源性。虽然重组的18 kb盒的单拷贝正确整合到了Agmo基因外显子2周围，但全基因组纳米孔测序显示Agmo基因座中有一个94 kb的重复，其中包含Agmo野生型外显子1-3。这种重复通过常规PCR误导了基因分型，但可以使用基于定量PCR的基因分型、靶向位点扩增测序和纳米孔测序来解决。尽管发生了这一事件，但该Agmo基因敲除小鼠模型缺乏AGMO酶活性，因此可用于研究其生理作用。
在同源重组的精确位点发生了重复事件，而常规的质量控制筛选（如荧光原位杂交或重组位点上的长距离PCR）未检测到该事件。纳米孔测序提供了一种经济便捷的方法来检测这种被低估的脱靶效应，表明其可用于对小鼠及其他模式生物的基因编辑进行额外的质量评估。
