Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-Cas9 technology offers powerful genome engineering capabilities.
• Controlling Cas9 guide RNA expression spatially and temporally is crucial for expanding its utility.
• Existing tRNA-based processing methods for guide RNA production suffer from high background noise.

Purpose of the Study:

• To develop a screening platform for optimizing tRNA variants for guide RNA expression.
• To engineer an improved tRNA scaffold for highly specific guide RNA production.
• To enable precise spatial and temporal control of CRISPR-based technologies.

Main Methods:

• Developed a screening platform for simultaneous measurement of promoter strength and 5'/3' processing efficiencies.
• Analyzed sequence determinants governing tRNA processing activities.
• Engineered a novel tRNA scaffold based on rational design principles.

Main Results:

• Identified dissociable sequence determinants for promoter strength and tRNA processing.
• Engineered a superior tRNA scaffold enabling highly specific guide RNA production from a Pol-II promoter.
• The new tRNA scaffold demonstrated performance equivalent to optimized Csy4-based systems.

Conclusions:

• The developed screening platform and engineered tRNA scaffold significantly improve guide RNA production specificity.
• This advancement offers enhanced control for CRISPR-based genome engineering applications.
• The findings contribute to both CRISPR technology development and fundamental tRNA biology research.