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

Akihiro Kuno,Arata Wakimoto,Atsushi Yoshiki,Kotaro Sakamoto,Yoshihisa Ikeda,Megumi Takemura,Tra Thi Huong Dinh,Kento Morimoto,Natsuki Mikami,Seiya Mizuno,Masafumi Muratani,Yuko Hamada,Satoru Takahashi,Yoko Daitoku,Fumihiro Sugiyama,Michito Hamada,Natsumi Iki,Yoko Tanimoto,Miyuki Ishida,Sayaka Suzuki ,Shin-Ichi Ayabe ,Katsuyuki Murata ,Kayoko Kato

doi:10.1371/journal.pbio.3001507

Akihiro Kuno, Arata Wakimoto + Show 21 more

Open Access

https://doi.org/10.1371/journal.pbio.3001507

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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.

Highlights

The development of new technologies such as CRISPR-Cas has facilitated genome editing of any species or cell type
Sniffles could not detect LoxP alleles (S2 File). These results provide evidence that DAJIN can accurately and comprehensively detect diverse mutations of floxed mice. Conventional approaches such as short-range PCR, Sanger sequencing, and PCR-RFLP are standard methods to detect on-target mutagenesis induced by CRISPR-Cas and other genome editing tools
We developed a genotyping method using a novel software, DAJIN, which can be applied for long-read sequencing to validate the quality of genome-edited organisms

Summary

Introduction

The development of new technologies such as CRISPR-Cas has facilitated genome editing of any species or cell type. Nucleases such as Cas and FokI and deaminase fused with Cas have sequencing data are available in the DDBJ DRA under accession number DRA011971 Been used to introduce DNA double-strand breaks and perform base editing, respectively [1,2,3]. As double-strand break repair pathways are regulated by host cells [4], verifying the result and selecting desired mutated alleles for precise genome editing are essential.

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Conclusion