3. Results
3.1 Ahomozygous strain of GOBPs deletion
A dual sgRNA-directed CRISPR-Cas9 system was employed to delete the GOBPs genes. According to the genomic arrangement of GOBP1 and GOBP2 (Figure 1), the sgRNA1/2 and sgRNA3/4 were designed to target the respective genes. The two specific sgRNAs and Cas9 protein were co-injected into yellow peach moth early embryos, respectively. In addition, to get both GOBP1 and GOBP2 (GOBP1/2) knockout homozygous population, four specific sgRNAs (sgRNA1/2/3/4) with Cas9 protein were using to co-inject. Among the injected eggs, 266 (96.72%), 180 (94.74%) and 199 (83.61%) neonates developed into adults (G0) for GOBP1, GOBP2, and GOBP1/2, respectively (Table 1). Based on the PCR amplification results, the primer pair G1/2-F/R revealed about 350 bp band (GOBP1) and about 600 bp band (GOBP2). Sequencing of those fragments confirmed a deletion event of the genes that were created and inherited in that single pair family. To get homozygous population, 10 homozygous males and females (1:1) were put together to produce the next generation (G1). Randomly extract DNA and sequenced using primer pair G1/2-F/R from the G1, results showed all the samples have only one expected band. To confirm those adults were homozygous, pair primers T1-F/R and T2-F/R were used for testing. The results showed wild type had one band, but not the same as knocked out GOBP1 and GOBP2 samples (Figure 1). Sequencing the fragments of GOBP1 and GOBP2 confirmed each gene had successfully knockout and the sequences consistent with our expected (Figure 1). All homozygous adults keep generation and can be used for further experiments.