Generating and testing reagents for CRISPR/Cas9 based homologous recombination and gene drive inTribolium

Abstract

AbstractCRISPR/Cas9 gene drive systems are possible in a few insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of gene drive systems is dependent upon direct experimentation. A critical aspect and potentially limiting factor of gene drive is the ability to induce Cas9-dependent homologous recombination. Here we report our attempts to induce Cas9-dependent homologous recombination and subsequent gene drive inTribolium castaneum. Utilizing constructs containing one or two target gRNAs in combination with Cas9 under two different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but a complete lack of evidence of homologous recombination and genetic drive. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of theTriboliumgenome, our results suggest thatTriboliumgenome may be refractory towards Cas9-induced homologous recombination and additional modifications will be necessary to increase the potential for homologous recombination.