CRISPR-Induced White Eyes in Tribolium Castaneum

White-eyed insects are nothing new; insects with normally dark eyes are known to have spontaneous mutants with white eyes. In insects with eyes solely pigmented by ommochromes, the metabolic pathway begins with a gene called vermilion that encodes the enzyme tryptophan 2,3-dioxygenase, and white-eyed insects also have been obtained through disruption of this gene. We explored the use of CRISPR to disrupt the vermilion gene in the red flour beetle, Tribolium castaneum, as a first step in transferring the technology to other stored product insects to produce white-eyed mutants. Guide RNA was targeted to T. castaneum vermilion, and newly-laid T. castaneum eggs were injected with a mixture of gRNA and Cas9 protein. Adult survivors had black eyes and were mated to a mutant strain of T. castaneum with white eyes, Tcv^w, in which the mutant vermilion gene contains a deletion of promoter and 80% of coding sequence. One CRISPR Go survivor mated to a Tcv^w individual had 19% white-eyed G1 progeny, and sequencing confirmed that there were multiple CRISPR events. Some of the G1 progeny with black eyes had a six base pair deletion around the gRNA site, which resulted in an in-frame deletion and a loss of two amino acids in the tryptophan 2,3-dioxygenase enzyme. However, sequencing also revealed that white-eyed G1 progeny had two base pair deletions causing a frameshift and insertion of a premature stop codon, with presumably no enzyme product. The distinction of the CRISPR allele (Tcv^CRw) and mutant allele (Tcv^w) in white-eyed G1 progeny was further confirmed via PCR using diagnostic primers specific for each allele. These data confirm the utility of the CRISPR system to produce eye color mutants in beetles for efficient markers in downstream germline transformation projects.