D3257: CRISPR/Cas9-mediated genome editing of a pigment transporter gene in the soybean looper, Chrysodeixis includens

Pigment transporter genes have been used as visible markers or classic genetic studies to create genetic control for destructive crops pests. CRISPR/Cas9 technology has significantly advanced research on non-model organisms, but it has not been successful in gene editing for the soybean looper, Chrysodeixis includens due to the limited genomic information and embryonic microinjection techniques. Here we report heritable knockout mutants in a pigment transporter gene, scarlet, in C. includens using CRISPR/Cas9-mediated mutagenesis. The scarlet locus identified in a genome assembly of C. includens consists of 14 exons and extends for 1,986 base pairs. Two small guide RNAs (sgRNAs) were designed to target the first exon of scarlet. Microinjection of Cas9 protein and two sgRNAs into the embryo successfully produced variable phenotypes with mutant eyes, accounting for about 16 % of mutation ratio. Genotype analysis revealed different indel mutations at the target site, resulting in a premature stop codon by frame shift. Single-pair mating of the mutant moths produced G1 offspring, and the homozygous mutant strain was established in G2. The mutant moths exhibited lightly pigmented compound eyes in both sexes, suggesting that scarlet may serve as a visible phenotypic marker for the tool. Our results provide the first successful case that the CRISPR/Cas9-mediated genome editing effectively induces mutations in C. includes, an economically important pest species in the United States.