Investigating the function of a unique DNA ligase (LIGK) in bdelloid rotifers

Abstract

Bdelloid rotifers are a class of microscopic invertebrates with an extraordinary DNA repair system that allows individuals to recover from DNA damage induced by desiccation or ionizing radiation. While this DNA repair system provides a potential means for surviving harsh conditions, the mechanism for DNA repair is not understood. Previous work determined that the gene encoding DNA ligase K (LIGK) is upregulated in the bdelloid Adineta vagafollowing exposure to ionizing radiation, suggesting a role for the LIGK protein in DNA repair. The objective of this project was to explore the role of LIGK during DNA repair in bdelloid rotifers by examining phylogenetic relationship of LIGK and other DNA ligases, and by identifying phenotypic effects when LIGKgene expression is repressed. Bioinformatics analyses were first done to uncover the evolutionary history of LIGK.Homologs of LIGKwere found sporadically in prokaryotic and eukaryotic lineages, but the gene is absent in related monogonont rotifers and most model organisms. The domain structure of LIGK proteins was distinct from canonical DNA ligases I, III and IV, consistent with a less well understood function for LIGK. Next, RNA interference (RNAi) and CRISPR/Cas9 genome editing were used to inhibit gene expression of LIGK. We first attempted using RNAi to silence LIGKexpression. We created an E. coli feeder strain that expressed a double stranded RNA copy of LIGK,and this was fed to rotifers at various concentrations and lengths of time. However, real‐time PCR failed to show strong evidence for a gene knockdown. We then used CRISPR/Cas9 genome editing to mutate and inactivate LIGK. We generated a single guide RNA (sgRNA) and used an in vitro Cas9 cleavage assay to demonstrate that sgRNA/Cas9 complexes cut the target sequence. We then designed a single‐stranded oligodeoxynucleotide (ssODN) template for homology‐directed repair that would introduce stop codons in the LIGKcoding sequence. Rotifer embryos were electroporated with sgRNA/Cas9 complexes and the ssODN. Then, individuals were screened by PCR using mutation‐specific primers which revealed evidence of genome editing and introduction of the desired mutation. To assess the effects of LIGKinactivation on DNA repair, we did desiccation recovery assays on RNAi‐ and CRISPR‐treated rotifers. If LIGKhas a role in DNA repair, then a lower recovery rate for treated rotifers compared to untreated or wild‐type rotifers would be expected. Overall, bioinformatics analysis and gene expression analyses are helping to understand the function of this unusual DNA ligase in bdelloid rotifers, which will broaden our understand of DNA repair beyond the typical model organisms.