D3192: Tribolium castaneum embryonic microinjection and inverse PCR

Tribolium castaneum is a model organism for other Coleoptera species due to their entire genome being available to analyze, their ability to be reared in a laboratory setting, as well as their relatively short generation time and high fecundity.  Cas9-based editing is invaluable in the field of genetic engineering and with editing anticipated to be significantly more efficient when expressed directly by the insect within the germline.  To accomplish this, a series of transposable element-based insertion were generated with the Cas9 protein under the control of the Vasa promoter; transgenic strains were identified by a GFP marker in the eyes with Cas9 expression documented by Western Blot analysis of both the somatic and germline tissue.  To verify the gene editing ability of each strain, we preformed microinjection of Cas9 T. casteneum embryos with synthetic guide RNA targeting the ebony gene, where a knockout results in a dark coloration of the adult cuticle.  In addition to screening for somatic mutagenesis, we also outcrossed injected G₀ offspring with a previously established ebony strain to identify germline mutations.  Inverse polymerase chain reaction (PCR) was also used to demonstrate the integration of the transgene within the genome.  Through this research we have found at least two Cas9-expressing strains of T. castaneum.