Engineering Guide RNAs for CRISPR-Based Biosensors

Area of Science:

• Biotechnology and Biosensing
• Molecular Biology and Genetics

Background:

• CRISPR-Cas systems offer programmable nucleic acid targeting for sensitive biosensor development.
• Native single-guide RNAs (sgRNAs) present limitations including instability, immunogenicity, and off-target effects, hindering clinical translation.

Purpose of the Study:

• To systematically review advancements in engineering single-guide RNAs (sgRNAs) for CRISPR-Cas biosensor applications.
• To address limitations of native sgRNAs and enhance biosensor performance for clinical diagnostics.

Main Methods:

• Review of CRISPR-Cas effector proteins, their mechanisms, and structural features.
• Analysis of native sgRNA limitations in biosensing.
• Examination of sgRNA engineering strategies: chemical modifications, structural remodeling, and functional integration.
• Integration of engineered sgRNAs into diverse biosensor platforms (e.g., microfluidics, wearables, POCT).

Main Results:

• sgRNA engineering strategies significantly improve biosensor stability, specificity, and reliability.
• Engineered sgRNAs have been integrated into various advanced biosensor platforms.
• Comparative analysis highlights performance metrics like detection signals and limits of detection.

Conclusions:

• Engineering sgRNAs is crucial for overcoming native limitations and enhancing CRISPR-based biosensor performance.
• Advances in sgRNA engineering accelerate the development of stable, specific, and reliable biosensors.
• Future directions focus on amplification-free, multiplexed, and clinically translatable CRISPR biosensors.