CRISPR/Cas9-mediated gene editing in an exogenous transgene and an endogenous sex determination gene in the Caribbean fruit fly, Anastrepha suspensa.
CRISPR/Cas9-mediated gene-editing, using injected Cas9 protein, was achieved in the Caribbean fruit fly, Anastrepha suspensa, by initially targeting an exogenous transgene, polyubiquitin-regulated EGFP (PUb-EGFP), for heritable non-homologous end-joining (NHEJ) knock-outs using an individual sgRNA. Multiple deletion mutations, ranging from two to five nts proximal to the target site, were identified phenotypically by the loss of green fluorescence in transgenic flies that were also marked with PUb-DsRed. This represented a relatively high efficiency rate of 29% for germ-line mutations. Similar conditions were then used to target an endogenous sex-determination gene, As-transformer-2 (Astra-2), using two sgRNAs that targeted independent exon sequences 671 bp apart. Somatic mutations were identified phenotypically in G0 adult flies at a frequency of 81% based upon intersexual genital morphology, expected to occur only in XX females since Astra-2 knock-outs by dsRNA do not have a phenotypic effect in XY males. Consistent with this expectation, twelve types of short indels, ranging from -15 nts to +5 nts, were identified proximal to the 5' sgRNA-1 target site in intersexual adults. However, the 3' sgRNA-2 target was only associated with a single 774 bp deletion extending from the sgRNA-1 target site to 100 bp downstream of the sgRNA-2 target. This is encouraging for the eventual use of dual target sites for homology-directed repair (HDR) insertions, but suggests that the sgRNA-2 target site tested may not be optimal for Astra-2 HDR modification.
['Li|Jianwei|J|', 'Handler|Alfred M|AM|']
D000818:Animals D064113:CRISPR-Cas Systems D059766:DNA End-Joining Repair D005260:Female D000072669:Gene Editing D017344:Genes, Insect D018095:Germ-Line Mutation D049452:Green Fluorescent Proteins D008297:Male D010641:Phenotype D017384:Sequence Deletion D033621:Tephritidae D019076:Transgenes
Sex determination CRISPR/Cas9 Insect pest management Tephritidae Transformer-2