Using Cas9 for Gene Editing in Tribolium Castaneum: What Do We Need to Make It Work?

The RNA-guided nuclease, Cas9, part of the bacterial adaptive immune system called CRISPR, has been hailed as a revolutionary tool for genome editing. As a result, it has been effectively deployed in several species to induce mutations in target genes, as well as insert genetic sequences of interest. The ability to identify and target any sequence of interest has made the CRISPR/Cas9 gene-editing system particularly attractive for research in species with limited resources, or for genomic stretches that have been resistant to other methods of study. However, outside Drosophila melanogaster, little has been done to study the efficacy of the system in insects. Understanding how Cas9 function may vary from species to species, from gene to gene, and even from sequence to sequence will be important for this tool to be effectively applied in novel ways. Recently, researchers published the successful use of Cas9 for gene editing in Tribolium castaneum. In our efforts to use Cas9 for dissection of the selfish genetic element, Medea, in this model organism, we encountered several issues that were not described by these researchers, inspiring us to pursue a more detailed look at the efficiency and limitations of gene editing in Tribolium. I will discuss some of our discoveries, including an analysis of the impact of target sequence on guide efficacy, and how the relative positions of guide targets affects the use of multiple guides.