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

Area of Science:

• Molecular Biology
• Genetics
• Entomology

Background:

• CRISPR/Cas9 technology is crucial for insect gene function studies.
• Low gene knockout efficiency in Hemiptera hinders research and genetic manipulation.
• Novel strategies are needed to enhance CRISPR/Cas9 efficacy in these species.

Purpose of the Study:

• To investigate methods for improving gene knockout efficiency in Hemiptera.
• To evaluate the impact of modified single-guide RNA (sgRNA) structures on knockout rates.
• To develop and test an enhanced CRISPR/Cas9 system for efficient gene editing in Hemiptera.

Main Methods:

• Modification of single-guide RNA (sgRNA) backbone structures.
• Utilizing in vitro and in vivo transcription methods for sgRNA.
• Implementing the PTG/Cas9 system and an integrated iPTG/Cas9 system.
• Assessing knockout efficiency through genetic analysis.

Main Results:

• Optimized sgRNA structures (Loop^5bp + M^T/C type) increased average knockout efficiency by 0.61-fold.
• The PTG/Cas9 system showed a 0.64-fold increase in knockout efficiency.
• The novel iPTG/Cas9 system, combining optimized sgRNA and PTG/Cas9, achieved a 1.45-fold increase in knockout efficiency.
• The iPTG/Cas9 system successfully enabled simultaneous knockout of two target sites and co-knockout of two genes.

Conclusions:

• Modified sgRNA structures and the PTG/Cas9 system significantly enhance CRISPR/Cas9 knockout efficiency in Hemiptera.
• The iPTG/Cas9 system offers a synergistic approach, markedly improving gene editing efficacy.
• This study introduces the first double knockout system in Hemiptera using iPTG/Cas9, providing a powerful tool for genetic research and pest control.