Identifying Prospective Genes That Function During DNA Repair in the Bdelloid Rotifer Adineta vaga

Abstract

The bdelloid rotifer Adineta vaga is a microinvertebrate with a highly efficient DNA repair system that allows individuals to recover from DNA damage caused by desiccation or ionizing radiation. The mechanism behind this remarkable DNA repair in A. vaga is not yet understood. Previous transcriptome sequencing in our lab has identified over 250 genes that are upregulated in response to DNA damage induced by ionizing radiation. These upregulated genes are the strongest candidates for having a role in DNA repair. The objective of this project is to characterize the functions of candidate genes involved in DNA repair in bdelloids and, thereby, establish potential pathways that comprise this mechanism. Three approaches were used to begin to investigate the functions of these candidate DNA repair genes in bdelloids. First, we used real‐time PCR to quantify gene expression of several candidate DNA repair genes during recovery from desiccation. Genes that were upregulated relative to the non‐desiccated control were identified as most likely to have a role in DNA repair as opposed to radiation resistance. Second, we constructed a yeast library of A. vaga proteins for use in yeast two‐hybrid screens against candidate DNA repair proteins. To produce the library, cDNAs representing the full complement of A. vaga proteins were cloned into plasmids that were then transformed into yeast. Yeast strains that each expressed a different candidate DNA repair protein would then be mated with the library to screen for protein‐protein interactions, which would provide evidence for related cellular functions. Third, we developed the molecular tools for using CRISPR/Cas9 genome editing to inactivate candidate genes in A. vaga. In vivo gene knockouts would provide mutant rotifers that could be studied to assess the roles of these genes in DNA repair. Single guide RNAs (sgRNAs) for several genes were created and incubated with the Cas9 enzyme to form sgRNA‐Cas9 complexes. These sgRNA‐Cas9 complexes were shown to successfully cleave their target gene sequences in vitro. To test this method in vivo, these sgRNA‐Cas9 complexes were transfected into A. vaga embryos via electroporation. Genomic DNA was purified from treated rotifers and used in T7 endonuclease I assays to identify subsequent mutations in the target genes. Once perfected, these methods of investigating the roles of candidate genes stand to elucidate the mechanism behind the extraordinary DNA repair in A. vaga.Support or Funding InformationFunding was provided by a Summer Undergraduate Research Fellowship from the Arkansas Department of Higher Education to JTW, and a Hendrix College Faculty Project Grant to AMS.