Highly efficient analysis of sgRNA and its impurities

Reliable analysis of single guide RNA, also known as sgRNA, is essential for ensuring high-quality results in CRISPR-Cas genome editing. Its analysis can be challenging, due to its relatively large size of approximately 100 nucleotides and the presence of closely related impurities such as n–1 or n+1 variants. This Application Note by Belgium-based Quality Assistance SA demonstrates an effective analytical approach using ion-pair reversed-phase HPLC in combination with the bioinert YMC Accura Triart Bio C18 column.

Bioinert hardware for reliable and reproducible oligonucleotide analysis

One key challenge in RNA analysis is the tendency of molecules to interact with metal surfaces, which can lead to adsorption effects, analyte loss, and carry-over. In addition, sgRNA can form complex secondary structures depending on temperature, salt concentration, and pH, which may influence chromatographic behaviour. The use of the bioinert coated YMC Accura column hardware helps to minimise unwanted interactions and ensures reliable and reproducible results.

Wide-pore phase for large biomolecules

The wide-pore stationary phase of the YMC Accura Triart Bio C18 column is specifically designed for large biomolecules such as oligonucleotides. This enables efficient mass transfer and results in improved peak shapes and high-resolution separations. The application data demonstrate clear separation of full-length sgRNA from closely related truncation impurities, even when these differ only slightly in length. 

Consistent performance and high sensitivity even at low concentrations

The method also shows consistent performance across a range of concentrations. High resolution is maintained even at lower sample concentrations, highlighting the sensitivity of the approach. Furthermore, blank runs confirm the absence of carry-over, underlining the effectiveness of the bioinert system in preventing non-specific interactions. This is particularly important for routine analysis workflows where reliability and reproducibility are essential.

High-resolution impurity profiling of sgRNA variants

Another important aspect is the ability to resolve multiple impurity species within a single analysis. The data show successful separation of several truncation impurities, including n–1, n–2, and n–5 species, as well as clear distinction from the full-length sgRNA. This level of resolution supports accurate impurity profiling and contributes to a deeper understanding of sample quality. 

Overall, this Application Note highlights a robust and reliable approach for sgRNA analysis. The combination of ion-pair reversed-phase HPLC and the wide-pore YMC Accura Triart Bio C18 column with bioinert hardware enables high-resolution separation, consistent performance, and reproducible results, supporting confident quality assessment in gene editing workflows.