Metabolic pathway interruption: CRISPR/Cas9-mediated knockout of tryptophan 2,3-dioxygenase in Tribolium castaneum

Abstract

The Tribolium castaneum vermilion gene encodes tryptophan 2,3-dioxygenase, a pivotal enzyme in the ommochrome pathway that is required for proper pigmentation of the eye. A white-eyed mutant strain of T. castaneum, vermilion^white (v^w), lacks eye pigmentation due to a deletion of unknown size that removes all but the 3'-end of the vermilion gene. To create a more defined mutation in vermilion, the CRISPR/Cas9-nuclease system was used to target wild type vermilion in preblastoderm T. castaneum embryos. As adults, all injected beetles had wild type (black) eye pigmentation; however, when outcrossed to v^w mates, one cross produced 19% white-eyed offspring. When the vermilion locus of these offspring was analyzed by target-site sequencing, it was determined that white-eyed individuals had a 2 bp deletion that resulted in a frame-shift mutation, presumably producing a nonfunctional enzyme. Interestingly, some of their black-eyed siblings also had a small deletion of 6 bp, but the resultant loss of two amino acids had no apparent impact on enzyme function. To establish a mutant strain homozygous for the CRISPR-induced knock-out allele, a CRISPR positive G₀ male was crossed to wild type females. Their progeny were self-crossed, and white-eyed progeny were used to establish the new strain. This mutant strain is herein named vermilion^ICE and will be used in future work in addition to or in place of v^w.

Keywords: CRISPR; Insect eye pigment; Ommochrome; Tribolium castaneum; Tryptophan 2,3-dioxygenase; Vermilion.