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

Abstract

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.

Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods

“…Therefore, we present a single analysis process applicable to a broad range of allele types (including point mutations, tags, conditional mutations and reporter alleles), which yields for each sample an alignment of all reads against the mutant sequence and a simplified and annotated alignment with only the reads that contain a sequence that is specific to the mutant. This analysis pipeline addresses a different question to that of in-depth characterisation of the composition of a mosaic genetic population, which would require computing-intensive phasing tools that do not rely on the assumption of a diploid genome [ 23 ]. It is therefore suited for screening and genotyping of edited animals or clonal cell populations, but would not apply to in-depth characterization of bulk cell culture experiments.…”

Universal method for generating knockout mice in multiple genetic backgrounds using zygote electroporation

“…Although this technology is comprehensive, its weakness is the high rate of sequencing errors. We overcame this bottleneck by adopting our novel software DAJIN, which can automatically identify and classify both intended and unintended diverse mutations (Kuno et al, 2022).…”

“…However, this evaluation cannot determine whether PCR bands reflect alleles with the intended deletion or unintended aberrant mutations. Therefore, we performed nanopore long-read sequencing and analysis using our software DAJIN (Kuno et al, 2022) to evaluate the presence of the intended alleles in each sample.…”

Convergent evolution of plant prickles is driven by repeated gene co-option over deep time

“…In addition, protein evidence from several published Solanaceae genomes ( 61, 62, 66 ), and the UniProt/SwissProt database were utilized to support gene annotation. Structural gene annotations were generated through the Mikado v2.0rc2 ( 67 ) framework, leveraging evidence from the Daijin pipeline ( 68 ). Additionally, microsynteny and orthology to Heinz v4.0 and Eggplant v4.0 were assessed using Microsynteny and Orthofinder v2.5.2 ( 69 ).…”