Abstract 1842 The Plausible Role of Histone H2A Proteins in Bdelloid Rotifer DNA Repair

Multiple DNA double-strand breaks caused by desiccation are lethal to most eukaryotes. Bdelloid rotifers, however, are an exception. These microscopic, aquatic animals possess unique DNA repair capabilities that allow them to survive desiccation. Their DNA repair mechanism is unusually efficient and not yet understood. In other eukaryotes, chromatin - specifically histone proteins - plays a key role in DNA repair. Bdelloids have seven histone H2A proteins with longer C-terminal tails than in other animals, so these proteins are good candidates for having a role in DNA repair. The objective of this project is to determine which bdelloid histone H2A proteins are likely involved in DNA repair. First, to identify polyadenylated histone mRNAs, 3' random amplification of cDNA ends (3' RACE) was used to sequence 3' UTRs of histone genes in the bdelloid Adineta vaga. Transcripts with poly-A tails likely represent histone variants that might regulate DNA repair. Next, the function of histone H2A proteins H2Abd and H2Abd1 in A. vaga were investigated by using CRISPR genome editing to inactivate the genes. Single-guide RNAs (sgRNAs) designed to excise each gene were first tested using an in vitro Cas9 cleavage assay. A DNA template was also designed to replace each histone gene with the GFP gene during homology-directed repair. CRISPR was carried out by electroporating rotifer embryos with the sgRNA/Cas9 complexes and the GFP repair template. Fluorescence microscopy was used to find evidence of genome editing via GFP expression in rotifers. Once a histone mutant is identified, CRISPR will be used to inactivate histone H2A genes that are potentially involved in DNA repair. Mutant rotifers will be desiccated to cause DNA damage, then rehydrated to induce DNA repair. Ultimately, a reduced recovery rate in mutant rotifers relative to wild type rotifers would suggest that the targeted histone H2A gene has an important role in DNA repair. Funding for this research was provided by a Morris and Ann Henry Hendrix College Odyssey Professorship to A. Schurko.