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

Single-stranded guide RNAs (sgRNAs) are important therapeutic modalities that facilitate selective genome editing by the CRISPR/Cas9 system. While these therapeutic modalities are synthesized through solid phase oligonucleotide synthesis similar to small interfering RNA (siRNAs) and antisense oligonucleotide (ASOs) therapeutics, their sequence length and complex secondary and tertiary structure hinder analytical characterization. The resulting current sgRNA methodologies have limited chromatographic selectivity near the FLP and limited MS compatibility. Here, we perform a systematic evaluation of chromatographic parameters to develop a highly selective ion-pairing reversed-phase chromatography separation for sgRNAs. We identify that stronger and more hydrophobic ion-pairing reagents promote the selectivity for long truncation impurities. Further, reduced flow rates and temperatures promote selectivity near the FLP. Together, this study reports the first single-nucleotide resolution chromatography method for an sgRNA modality while employing a highly MS-compatible mobile phase. This work provides further evidence that the sgRNA chromatographic selectivity is highly independent of on-column mass transfer. This methodology will enable the high-resolution characterization of sgRNA therapeutics, providing further insights into impurity profiles to facilitate toxicological studies and process development of these genetic medicines.