Establishing a chimeric tRNA-sgRNA scaffold and computational basis for enhanced CRISPR interference.

Area of Science:

• Molecular Biology
• Genetic Engineering
• Biotechnology

Background:

• The CRISPR/Cas9 system is a powerful genome engineering tool, but its efficacy depends on single-guide RNA (sgRNA) stability and expression.
• Transfer RNAs (tRNAs) possess inherent stability, offering a potential scaffold to improve sgRNA performance.

Purpose of the Study:

• To develop and evaluate a chimeric tRNA-sgRNA (tgRNA) construct for enhanced gene regulation.
• To assess the stability and gene knockdown efficiency of the novel tgRNA compared to conventional sgRNAs.

Main Methods:

• Integrated sgRNA into a tRNA scaffold (Sephadex aptamer-human HBV ε tRNA) to create a chimeric tgRNA (SeptgRNA).
• Applied SeptgRNA with deactivated Cas9 (dCas9) for CRISPR interference (CRISPRi) targeting E. coli ampC and ompA genes.
• Utilized molecular docking and dynamics simulations to analyze complex stability.

Main Results:

• SeptgRNA showed significantly higher accumulation than conventional sgRNAs in E. coli.
• CRISPRi using SeptgRNA and dCas9 achieved superior gene expression suppression compared to sgRNA-based CRISPRi.
• Computational simulations confirmed SeptRNA scaffold enhances sgRNA stability and dCas9-tgRNA-DNA complex integrity.

Conclusions:

• Chimeric tgRNAs represent a viable strategy for improving gene knockdown via enhanced stability and expression.
• This study advances the CRISPR/Cas9 toolkit, demonstrating the potential of tRNA scaffolds in genetic engineering.