sgRNA structure optimization and PTG/Cas9 system synergistically boost gene knockout efficiency in an insect

Knockouts mediated by CRISPR/Cas9 technology are widely used to study insect gene functions, but the efficiency in Hemiptera is low. New strategies are urgently needed to improve gene knockout efficiency. This study initially explored the impact of modifying the fundamental backbone structure of single guide RNA (sgRNA) on knockout efficiency. The results indicated that both in vitro and in vivo transcription of sgRNA structures (Loop5bp + MT/C type) increased average knockout efficiency by 0.61-fold compared to the original sgRNA. In addition, the PTG/Cas9 system was observed to induce a 0.64-fold increase in average knockout efficiency using the original sgRNA. Notably, an integrated PTG/Cas9 system (iPTG/Cas9 system), the integration of optimized sgRNA structures (Loop5bp + MT/C type) into the conventional PTG/Cas9 system, demonstrated a synergistic effect, resulting in a 1.45-fold increase in average knockout efficiency compared to the original sgRNA structure. The iPTG/Cas9 system was effectively used to simultaneously knockout two different target sites within a single gene and to co-knockout two genes. This study represents the first application of the iPTG/Cas9 system to establish a double knockout system in Hemiptera, offering a promising approach to enhance knockout efficiency in species with low efficiency and improve genetic manipulation tools for pest control.