DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

PLoS Biol. 2022 Jan 18;20(1):e3001507. doi: 10.1371/journal.pbio.3001507. eCollection 2022 Jan.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Gene Editing*
  • Gene Knock-In Techniques
  • Genome
  • Genotype
  • Genotyping Techniques / methods*
  • INDEL Mutation
  • Machine Learning
  • Mice, Inbred C57BL
  • Mice, Inbred ICR
  • Mutation
  • Nanopore Sequencing
  • Sequence Analysis, DNA
  • Software*

Grants and funding

Grant number 19H03142 to S.M. and A.K. from the Ministry of Education, Culture, Sports, Science, and Technology. Grant number 20ae0201011h0003 to S.M. and S.T. from the Japan Agency for Medical Research and Development. Grant number JPMJPF2017 to S.T. and A.Y. from the Japan Science and Technology Agency. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.