Sequence Mapping of sgRNA Digests: Leveraging Xevo™ MRT Mass Spectrometer Performance and Streamlining Data Analysis with MAP Sequence 2.0

Importance of the Topic

Single-guide RNAs (sgRNAs) are essential components of CRISPR-Cas9 gene editing, directing the nuclease to specific DNA targets. Accurate and comprehensive sequence mapping of sgRNAs is critical for ensuring their integrity, confirming chemical modifications, and supporting regulatory compliance in research, diagnostics, and therapeutic applications.

Objectives and Study Overview

• Develop a compliance-ready workflow for automated sequence mapping of digested sgRNAs.
• Apply multiple ribonucleases (RNase T1, RapiZyme™ MC1, RapiZyme™ Cusativin) to maximize sequence coverage.
• Acquire UPLC-MSE data on the Xevo™ MRT QTOF Mass Spectrometer.
• Streamline data analysis using waters_connect™ MAP Sequence Application v2.0.

Methodology and Instrumentation

• Sample Preparation: Waters 100-mer sgRNA standard with phosphorothioate 2′-O-Me modifications was digested separately with RNase T1 (15 min, 37 °C), RapiZyme MC1 (30 min, 30 °C, pH 8.0) and RapiZyme Cusativin (30 min, 30 °C, pH 9.0).
• UPLC Conditions: ACQUITY™ Premier UPLC with BEH C18 column (2.1×150 mm, 1.7 µm) at 70 °C, gradient of DIPEA/HFIP aqueous and acetonitrile solvents, 400 µL/min flow, 5 µL injection.
• Mass Spectrometry: Xevo™ MRT QTOF, ESI negative mode, MSE acquisition (low- and high-energy channels), 2 Hz scan rate, sub-ppm mass accuracy, resolving power ~100 000.
• Data Analysis: waters_connect™ Platform v4.1 with SYNTHETIC Library App v2.0 for sequence entry and in-silico digestion, MAP Sequence App v2.0 for automated peak assignment, fragment validation, and coverage visualization.

Main Results and Discussion

• Automated processing matched precursors and fragments to in-silico predictions with average RMS mass accuracy < 1 ppm.
• High-resolution data resolved isotopic envelopes across multiple charge states (–2 to –8) and confirmed elution profiles.
• Individual digests yielded up to 91 % coverage (RapiZyme MC1) and 100 % coverage (RapiZyme Cusativin) due to unique cleavage specificities and controlled missed cleavages.
• Combined mapping from all three enzymes achieved complete (100 %) sequence coverage of the sgRNA standard.

Benefits and Practical Applications

• Streamlined, compliance-ready informatics workflow reduces manual intervention and accelerates analysis.
• High confidence in modification detection and sequence verification supports QA/QC in RNA therapeutics manufacturing.
• Adaptable to diverse oligonucleotide and mRNA applications, enabling robust impurity profiling and structural studies.

Future Trends and Potential Applications

• Integration of additional ribonucleases and digestion strategies for more complex RNA constructs.
• Enhanced bioinformatics tools for real-time quality control in high-throughput production.
• Application to large or highly modified RNAs, including therapeutic mRNAs and siRNAs.
• Coupling with emerging mass spectrometry technologies for even greater sensitivity and speed.

Conclusion

The described workflow combining multi-enzyme digestion, Xevo™ MRT QTOF MS, and MAP Sequence App v2.0 delivers complete and reliable sgRNA sequence mapping with sub-ppm accuracy. This approach enhances confidence in RNA characterization, streamlines regulatory compliance, and can be extended to a wide range of oligonucleotide analyses.

Reference

1. Jinek M et al. Science. 2012;337:816–821.
2. Jiang F, Doudna JA. Annu Rev Biophys. 2017;46:505–529.
3. Ganbaatar U, Liu C. Front Cell Infect Microbiol. 2021;11:663949.
4. Waters application note. LC-MS Analysis of siRNA, Single Guide RNA and Impurities. 2022;720007546.
5. Goyon A et al. Anal Chem. 2022;93:14792–14801.
6. Waters application note. RNA Digestion Product Mapping Using UPLC-MS. 2024;720008553.
7. Waters application note. Tunable Digestion of RNA Using RapiZyme RNases. 2024;720008539.
8. Waters application note. Oligo Mapping of mRNA Digests. 2025;720008677.
9. Waters application note. Analysis of mRNA Cap Impurity Profiles. 2025;720008793.
10. Grunberg S et al. Protein Expr Purif. 2022;190:105987.
11. Thakur P et al. Int J Mol Sci. 2022;23:7021.