D3046: Transgene elimination through utilization of the single-strand annealing dsDNA repair pathway in the model organism Tribolium castaneum

Monday, November 11, 2024

9:00 AM – 5:00 PM MST

Location: Phoenix Convention Center, Exhibit Hall 6, PCC

Presenting Author(s)

CV

Collin Valentin (he/him/his)

Graduate Student
Texas A&M University
College Station, Texas

Co-Author(s)

MI

Musa Isah

Texas A&M University
College Station, Texas
ZA

Zach N. Adelman

Professor
Texas A&M University
College Station, Texas

The release of transgenic organisms shows great potential as a means of addressing the agricultural and medical concerns associated with pest insect species. Gene drive systems are hypothesized to be an efficient means to spread a transgene through a population above Mendelian rates, but removal of that transgene is not an inherent part of these systems. Here we outline one means to allow for this removal in the model organism Tribolium castaneum based upon previous work from our lab in Aedes aegypti, through utilization of the single-strand annealing (SSA) dsDNA repair pathway. To test this, we have utilized embryonic microinjection to create two transgenic SSA-reporter T. castaneum strains containing the recognition site for the homing endonuclease I-Ani I flanked by 750bp and 200bp direct repeat sequences, respectively. Both reporters were integrated into an Ebony knockout background to allow for easy visualization of transgene removal by scoring a reversion to wild-type pigmentation. We have also developed two T. castaneum strains which endogenously express I-Ani I under the control of either the polyubiquitin or nanos genes, and are further developing strains which utilize the germ-line specific promoters Cyclin B3 and Fizzy. Crossing SSA reporter strains with the I-Ani I expressing strains is expected to result in double-stand break formation followed by SSA repair, excising the transgene and restoring a wild type phenotype. If successful, this system could contribute to progress towards a gene drive system that could responsibly leave a laboratory setting and promote further study surrounding their practical applications.