sgRNA Single-Nucleotide Resolution by Ion-Pairing Reversed-Phase Chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Single-stranded guide RNAs (sgRNAs) are crucial for CRISPR/Cas9 genome editing therapies.
• Current analytical methods for sgRNAs, including chromatography, lack sufficient selectivity and mass spectrometry (MS) compatibility.
• The complex structure of sgRNAs complicates their characterization, hindering therapeutic development.

Purpose of the Study:

• To develop a highly selective ion-pairing reversed-phase chromatography (IP-RPC) method for sgRNA analysis.
• To overcome limitations in existing chromatographic techniques for sgRNA characterization.
• To enable high-resolution analysis of sgRNA therapeutics for improved process development and toxicological studies.

Main Methods:

• Systematic evaluation of chromatographic parameters for sgRNA separation.
• Optimization of ion-pairing reagents, flow rates, and temperatures for IP-RPC.
• Development of an MS-compatible mobile phase for enhanced analysis.

Main Results:

• Achieved the first single-nucleotide resolution chromatography method for sgRNAs.
• Demonstrated that stronger, more hydrophobic ion-pairing reagents enhance selectivity for truncation impurities.
• Identified that reduced flow rates and temperatures improve selectivity near the FLP (presumably Full-Length Product).

Conclusions:

• The developed IP-RPC method provides high-resolution characterization of sgRNA therapeutics.
• This methodology facilitates insights into sgRNA impurity profiles, crucial for toxicological assessments.
• The findings support the advancement of sgRNA-based genetic medicines through improved process development and quality control.