Knockout of GST Cluster Using CRISPR/Cas9

To further analyze the function of the GST cluster, we knocked out the GST cluster using the CRISPR/Cas9 system. A pair of sgRNAs were designed using the sgRNAcas9 design tool (Supplementary Table 1)^{62 (link)}. sgRNA1 and sgRNA2 were located on GST_116 and GST_121, respectively. The sgRNA target sequences were checked in a search of the H. armigera genome using the sgRNAcas9 design tool, and no potential off-target sites were identified. A PCR-based approach was used to prepare sgRNA according to the manufacturer’s instructions (GeneArt Precision gRNA Synthesis Kit, Thermo Fisher Scientific, USA). The template DNA for the in vitro transcription of the sgRNAs was made using PCR-based fusion of two oligonucleotides with the T7 promoter (Target F: TAATACGACTCACTATAG + the target sequence; Target R: TTCTAGCTCTAAAAC + the reverse complementary sequence of the target). The PCR conditions were as reported by Jin et al.^{35 (link)}. The sgRNAs were synthesized by in vitro transcription using the GeneArt Precision gRNA Synthesis Kit, according to the manufacturer’s instructions. The Cas9 protein (GeneArt Platinum Cas9 Nuclease) was purchased from Thermo Fisher Scientific (Shanghai, China).