Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Summary:The determination of S-(l,2-dicarboxyethyl)glutathione and reduced glutathione (GSH) in the rabbit lens and liver was developed using an isotachophoretic analyser.The recovery of S-(l,2-dicarboxyethyl)GSH from the rabbit liver after ion-exchange treatment was 96.8 ± 11.3% (n = 3). The contents of S-(l,2-dicarboxyethyl)GSH in the rabbit lens and liver were 219.9 ± 29.1 (n = 5) and 44.0 ± 13.5 (n = 8) nmol/g, respectively.The contents of S-(l,2-dicarboxyethyl)GSH in the lens and GSH in the lens and liver of naphthalene-treated rabbits was also determined by this method 24 hours after naphthalene administration, at which time the axial opacity "spichen" was observed at the equatorial region of the lens. The content of S-(l,2-dicarboxyethyl)GSH in the lens decreased in proportion to the content of GSH.During the further development of true lens opacity after naphthalene administration, the S-(l,2-dicarboxyethyl)GSH content further compared with that in the spichen stage, but the S-(l,2-dicarboxyethyl)GSH content of the lens that did not develop true opacity after naphthalene administration returned to the normal level. The change of S-(l,2-dicarboxyethyl)GSH content of the lens in the spichen and true opacity stages coincided with that of GSH content.On the other hand, the content of GSH of the liver decreased markedly until 24 hours after naphthalene administration, then returned to normal, irrespective of whether true opacity did or did not subsequently develop.

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Genome editing induces various on-target mutations. Accurate identification of mutations in founder mice and cell clones is essential to perform reliable genome editing experiments. However, no genotyping method allows the comprehensive analysis of diverse mutations. We developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify diverse mutations, including point mutations, deletions, inversions, and knock-in. Our genotyping method with DAJIN can handle approximately 100 samples within a day and may become a new standard for validating genome editing outcomes.