Expanding the Toolkit for Genome Editing in a Disease Vector, Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis.

CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, , a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, (), and the juvenile hormone receptor, (). PiggyBac transformation vectors were prepared to express sgRNAs targeting and under the control of the U6 promoter. Transgenic expressing -sgRNA or -sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in -sgRNA and -sgRNA groups, respectively, showed mutations in their target genes.