2.2 Intron detection and preparation of guide-RNAs
Due to the lack of genome database of the yellow peach moth, firstly we have to design primers based on the cDNA sequence of GOBPs, using the DNA as a template for PCR then sequencing to determine its intron sequence. According to some previous research (Zhang and Reed, 2016; Wang et al., 2020a), two Cas9 cut sites were designed for producing long deletions in GOBP1 and GOBP2 target loci. sgRNAs of GOBP1 and GOBP2 were designed by manually searching genomic regions for GGN18NGG or N20NGG protospacer adjacent motif (PAM) sequences on the sense or antisense strands (Figure 1 and Supplementary Table S1). sgRNA template was transcribed in vitrowith the specific oligonucleotide encoding T7 polymerase-binding site and the sgRNA target sequences following the manufacturer’s instruction of GeneArtTM Precision gRNA Synthesis Kit (Thermo Fisher Scientific, USA).