Efficient collection of a large number of mutations by mutagenesis of DNA damage response defective animals

Yuji Suehiro,Shohei Mitani,Tomoko Motohashi,Katsufumi Dejima,Satoru Iwata,Sawako Yoshina

doi:10.1038/s41598-021-87226-7

Yuji Suehiro, Shohei Mitani + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-87226-7

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Abstract

With the development of massive parallel sequencing technology, it has become easier to establish new model organisms that are ideally suited to the specific biological phenomena of interest. Considering the history of research using classical model organisms, we believe that the efficient construction and sharing of gene mutation libraries will facilitate the progress of studies using these new model organisms. Using C. elegans, we applied the TMP/UV mutagenesis method to animals lacking function in the DNA damage response genes atm-1 and xpc-1. This method produces genetic mutations three times more efficiently than mutagenesis of wild-type animals. Furthermore, we confirmed that the use of next-generation sequencing and the elimination of false positives through machine learning could automate the process of mutation identification with an accuracy of over 95%. Eventually, we sequenced the whole genomes of 488 strains and isolated 981 novel mutations generated by the present method; these strains have been made available to anyone who wants to use them. Since the targeted DNA damage response genes are well conserved and the mutagens used in this study are also effective in a variety of species, we believe that our method is generally applicable to a wide range of animal species.

Highlights

With the development of massive parallel sequencing technology, it has become easier to establish new model organisms that are ideally suited to the specific biological phenomena of interest
DNA monoadducts are repaired by two major nucleotide excision repair (NER) pathways, and interstrand crosslinks (ICLs) are repaired by the Fanconi anemia pathway, NER, and homologous recombination (HR) 21–23
Since the loss of function of DNA damage response (DDR)-related genes leads to the accumulation of mutations in C. elegans[27], we hypothesized that the trimethyl psoralen and ultraviolet (TMP/UV) treatment of DDR mutants leads to efficient isolation of mutant strains

Summary

Introduction

With the development of massive parallel sequencing technology, it has become easier to establish new model organisms that are ideally suited to the specific biological phenomena of interest. Using C. elegans, we applied the TMP/UV mutagenesis method to animals lacking function in the DNA damage response genes atm-1 and xpc-1. Of the effort required to isolate individual mutations.to efficiently construct mutant libraries for the analysis of individual gene functions, it is important to establish methods for the generation of organisms with mutations that can be genotyped with high frequency to the extent that they can be isolated by crossing. We report that TMP/UV treatment of XPC and ATM double mutants and subsequent bioinformatics analysis by WGS and machine learning allows us to isolate new mutant lines three times more efficiently than mutagenesis of wild-type animals. We isolated 981 new mutant strains, which we are distributing to the research community

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