Elucidating the Mechanism of DNA Repair in Bdelloid Rotifers by Using RNAi Knockdown and CRISPR/Cas9 Editing

Abstract

Genomic DNA regularly becomes damaged by numerous ways, including desiccation, radiation exposure, and during recombination in cell division. Fortunately, cells have DNA repair pathways that seek out and correct damage. The bdelloid rotifer Adineta vaga is an excellent model organism to study DNA repair because it has a highly efficient DNA repair system. Bdelloids can recover from desiccation and return to life by repairing DNA damage endured in the desiccated state. Bdelloids have also been shown to recover from DNA damage caused by massive doses of ionizing radiation. The mechanisms of this miraculous DNA repair in these aquatic invertebrates are unknown. This project explores using RNA interference (RNAi) and CRISPR/Cas9 genome editing as tools to investigate the function of DNA repair genes, MSH6 and MLH1, in bdelloid rotifers. When target genes are knocked down or inactivated, DNA repair in bdelloids should be compromised. For RNAi, two methods were explored. For the “feeding method,” bdelloids were fed strains of E. coli that express double stranded RNA (dsRNA) copies of MLH1 and MSH6. For the “soaking method,” dsRNA copies of MLH1 and MSH6 were synthesized, and bdelloids were transfected with these molecules. The expectation was that ingested or transfected dsRNA would induce RNAi in bdelloid rotifers. Real‐time PCR followed to quantify expression of the target gene and determine if the RNAi knockdown was successful. For the feeding method, an MLH1 knockdown was observed 6–12 hours after feeding, but the effect was absent 24 hours after feeding. For the soaking method, an MSH6 knockdown was only present when rotifers were transfected with 0.2 μg dsRNA and incubated for 48 hours. As an alternative tool for studying gene function, CRISPR/Cas9 genome editing was done to mutate DNA repair genes. For utilizing CRISPR/Cas9, a single guide RNA that targets MLH1 was synthesized and used in a Cas9 in vitro cleavage assay to determine its ability to cut the target sequence. The guide RNA was then combined with Cas9, and these ribonucleoprotein complexes were electroporated into bdelloids. Mutation detection using T7 endonuclease I was then done to identify alterations to the gene sequence. Gene knockdowns using RNAi and gene mutations using CRISPR/Cas9 will provide a reverse genetics approach to identify genes that have a role in bdelloid DNA repair. This could enhance our understanding of similar mechanisms in traditional model systems, which could have implications for biomedical research.Support or Funding InformationThis work was supported by grants from the Hendrix College Odyssey Program and the Hugh Stevens Moseley Undergraduate Research Awards.