Abstract 1908 Using CRISPR to Inactivate Candidate DNA Repair Genes in Bdelloid Rotifers

Bdelloid rotifers are microscopic invertebrates that reside in freshwater environments. These creatures possess the ability to survive desiccation not by preventing DNA damage, like in many eukaryotes, but instead by repairing DNA damage caused by desiccation. However, the mechanism for this repair is unknown. Identifying candidate genes and proteins that regulate DNA repair is vital to understanding this process. Furthermore, previous work identified a homolog of prokaryotic DNA ligase E (LigE) in the bdelloid Adineta vaga that is upregulated following DNA damage. The objective of this project was to use CRISPR genome editing to inactivate the LigE gene in the bdelloid A. vaga to investigate its role in DNA repair. To achieve this, two sgRNAs were designed to excise the LigE and GAPDH (as a control) from the genome. In vitro Cas9 cleavage assays were used to determine the efficiency of the sgRNAs. DNA templates were designed to replace the target genes during homology-directed repair. For CRISPR in A. vaga, different electroporation conditions using GFP- tagged Cas9 were explored.Then, CRISPR was carried out by electroporating embryos with the sgRNA/ Cas9 complexes and GFP repair template. For evidence of genome editing, rotifers were examined under a fluorescence microscope for GFP expression. We also used real-time PCR and deep amplicon sequencing to screen embryos for mutations. Once a mutant rotifer with the inactivated LigE gene is identified, individuals will be desiccated and subsequently rehydrated to induce DNA repair. The recovery rate for desiccated wild-type rotifers at varying time stamps was determinedin this project. Thus, a reduced rate of recovery in mutant rotifers compared to the wild-type would insinuate the LigE gene plays a crucial role in DNA repair. Funding for this research was provided by a Morris and Ann Henry Hendrix College Odyssey Professorship to A. Schurko.