Optimizing IP-RP CRISPR sgRNA Purification Passes With High Efficiency Oligonucleotide Certified BEH 300 Å C18 5 μm Preparative Sorbent

Importance of the Topic

CRISPR technology relies on single-guide RNA (sgRNA) to direct precise gene editing and epigenetic modifications. High-purity sgRNA is essential to avoid off-target effects and ensure therapeutic precision. Optimizing purification methods enhances the safety and efficacy of CRISPR applications.

Objectives and Study Overview

This study evaluates the purification of a 100-mer sgRNA using ion-pairing reverse-phase liquid chromatography (IP-RP LC). It compares traditional large-particle sorbents to a newly commercialized, batch-tested 5 µm BEH 300 Å C18 preparative column. The aim is to assess improvements in impurity separation, retention efficiency and loading capacity.

Methodology

A 100-mer sgRNA with 2ʹ-O-methyl and phosphorothioate modifications was prepared by solid-phase synthesis. Initial screening gradients in hexylammonium acetate (HAA) buffers determined elution times, followed by tailored gradients targeting the full-length product. Preparative runs used 100 mM HAA (pH 7.0) as mobile phase A and 50 mM HAA in 50 % acetonitrile (pH 7.0) as mobile phase B at 20 mL/min. Analytical purity was confirmed by UPLC-UV/MS using triethylamine (TEA) and hexafluoroisopropanol (HFIP) buffers at elevated temperature.

Instrumentation

• Preparative system: Waters LC Prep 150 with 2545 Quaternary Solvent Manager, 2489 UV/Vis Detector and WFC III; data acquired in MassLynx
• Preparative columns: XBridge Peptide BEH C18 OBD 300 Å 10 µm (19×250 mm), XBridge Oligonucleotide BEH C18 OBD 300 Å 5 µm (19×150 mm), and 8 µm PS-DVB 300 Å polymeric column (25×150 mm)
• Analytical system: Waters ACQUITY UPLC H-Class Plus with BioQuaternary Solvent Manager, Bio Sample Manager FTN-H, ACQUITY Premier tunable UV, Empower software; ACQUITY Premier Oligo BEH C18 300 Å 1.7 µm (2.1×100 mm) column at 80 °C

Key Results and Discussion

The 5 µm BEH 300 Å C18 column outperformed the 10 µm sorbent, raising purity from ~35 % to 41–45 % and reducing front-end impurities from ~16 % to ~11 %. Enhanced surface area improved binding and separation, supporting higher sample loads without co-elution. In contrast, the 8 µm PS-DVB column showed limited impurity removal (~34–37 % early impurities) and pressure instability.

Benefits and Practical Applications

• Higher resolution separation of full-length sgRNA from truncated and modified by-products
• Improved loading capacity for preparative runs, increasing throughput
• Enhanced column stability and reproducibility for multiple purification cycles
• Reduction of off-target risk through higher sgRNA purity

Future Trends and Potential Applications

Addressing late-eluting impurities will require optimization of sgRNA synthesis and deprotection steps. Implementation of concave gradient designs, coupling weak and strong ion pairing, and integrating anion-exchange steps may further refine purification quality and efficiency.

Conclusion

The batch-tested XBridge Oligonucleotide BEH C18 300 Å 5 µm preparative column demonstrates significant advantages in impurity separation and loading capacity for sgRNA purification. Its superior performance makes it a preferred choice for high-precision CRISPR applications and preparative oligonucleotide separations.

References

• Villiger L, Joung J, Koblan L et al. CRISPR technologies for genome, epigenome and transcriptome editing. Nat Rev Mol Cell Biol. 2024;25:464–487.
• Nakamura M, Gao Y, Dominguez AA et al. CRISPR technologies for precise epigenome editing. Nat Cell Biol. 2021;23:11–22.
• Minkner R, Boonyakida J, Park EY, Watzig H. Oligonucleotide separation techniques for purification and analysis: what can we learn for today’s tasks? Electrophoresis. 2022;43:2402–2427.