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).