Hendrix College Little Rock, Arkansas, United States
Mitchell Rotenberry (Hendrix College)| Andrew Schurko (Hendrix College)
Bdelloid rotifers are aquatic microinvertebrates with an efficient DNA repair system that allows them to recover from severe DNA damage caused by desiccation. While the underlying mechanisms of the DNA repair process in bdelloids are unknown, we have identified several candidate genes that possibly play a role. The objective of this project was to develop a method for using CRISPR/Cas9 genome editing as a tool for investigating the functions of candidate DNA repair genes in the bdelloid Adineta vaga. First, a single guide RNA (sgRNA) that targets the DNA Polymerase β (POLB) gene was synthesized. As a pre-validation step to determine the cleavage efficiency of the sgRNA, the sgRNA was incubated in vitro with the target gene and the Cas9 enzyme. Cleavage products were then analyzed by gel electrophoresis. To use the sgRNA for CRISPR/Cas9 genome editing of POLB in A. vaga, we first designed a single-stranded oligonucleotide donor (ssODN) to be used as the template for homology-directed repair. The 100-nucleotide ssODN was predicted to introduce a stop codon in POLB resulting in a truncated protein if integrated into the genome by homology-directed repair. Next, we carried out in vivo genome editing in A. vaga by electroporating embryos with sgRNA/Cas9 ribonucleoprotein complexes and the ssODN with a variety of concentrations of each component. To identify individuals that had undergone genome editing and contain an inactivated POLB gene, we used a combination of the Surveyor nuclease (for detecting mismatches) and PCR with mutation-specific primers. In subsequent assays, individuals with CRISPR-induced POLB mutations will be desiccated and their recovery rates can be quantified relative to a wild-type. The expectation is that a reduced recovery rate in mutants will correspond with the POLB gene being relevant in DNA repair. Understanding this mechanism of DNA repair will allow for a more generalized understanding beyond model organisms and could have implications for biomedical research.
Support or Funding Information
This work was supported by a Summer Undergraduate Research Fellowship from the Arkansas Department of Higher Education to Mitchell Rotenberry.
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