Sequence Mapping of sgRNA by RNase T1 Digestion and LC/Q-TOF Analysis

Significance of the topic

The accurate sequence confirmation of single guide RNA (sgRNA) is critical for development, manufacturing and quality control of CRISPR‑Cas9 reagents. Sequence errors, truncations or unintended chemical modifications can reduce targeting fidelity and editing performance. The described LC/Q‑TOF RNase digestion workflow provides a practical route to sequence-level mapping that is simpler and more robust than attempting full-length tandem MS/MS for long, modified RNA species.

Objectives and overview of the study

The study aimed to demonstrate a reproducible analytical workflow for sgRNA sequence confirmation by: (1) verifying intact molecular mass of a 100‑nt, terminally modified sgRNA; (2) generating RNase T1 digestion fragments; (3) separating fragments by ion‑pair reversed‑phase LC; and (4) assigning fragment identities by high‑resolution accurate mass LC/Q‑TOF to produce a sequence coverage map suitable for QC purposes.

Methodology

Sample preparation and digestion:

• Starting material: commercially synthesized 100‑mer sgRNA with terminal 2′‑O‑methyl nucleotides and phosphorothioate linkages at both ends.
• Sample concentration: diluted to 1 pmol/µL in 50 mM Tris‑HCl, 1 mM EDTA, pH 7.5.
• RNase T1 digestion: enzyme added at manufacturer recommended ratio (≈0.2 µL enzyme per µg RNA), incubated 30 min at 37 °C. After digestion, aliquots were directly injected for LC/MS.

Analytical strategy:

• Intact LC/MS was used to confirm molecular weight of the full‑length sgRNA.
• Bottom‑up sequence mapping: RNase T1 cleaves 3′ of guanosine residues to produce guanosine‑terminated fragments (Gp‑products). Fragment masses and retention times were matched to expected cleavage products to build a sequence map.

Used instrumentation

The analyses combined ion‑pair reversed‑phase LC and high‑resolution Q‑TOF MS:

• LC system: Agilent 1290 Infinity II Bio LC with high‑speed pump, multisampler (4 °C thermostat) and multicolumn thermostat (column at 50 °C).
• Column: Agilent Altura Oligo HPH (2.1 × 150 mm, 2.7 µm) featuring Ultra Inert surface to minimize metal‑mediated oligonucleotide adsorption.
• Mobile phases: aqueous solvent with 15 mM hexylamine (HA) and 25 mM HFIP and acetonitrile as organic modifier; flow rate 0.3 mL/min; gradient optimized for oligonucleotide separation; injection volume 5 µL.
• MS system: Agilent 6545XT AdvanceBio LC/Q‑TOF with Dual Jet Stream ESI (negative polarity). Acquisition: MS scan mode, m/z 500–3,000, ~2 spectra/s, Extended Dynamic Range tuning; key source/settings included gas temperatures 300–350 °C, capillary ~4,500 V, fragmentor 175 V, skimmer 65 V.
• Software: Agilent MassHunter and BioConfirm for acquisition and spectral deconvolution.

Main results and discussion

Intact mass analysis:

• Deconvoluted intact mass observed: 32,367.9478 Da versus theoretical average mass 32,367.6992 Da (Δ ≈ 0.2486 Da), confirming expected full‑length mass within instrument performance.

RNase T1 digest mapping:

• RNase T1 cleavage theoretically yields 25 products for the 24 G residues; however, many digestion products are sequence‑non‑unique (identical short sequences at multiple positions).
• Chromatographic separation on the Altura Oligo HPH produced well‑resolved peaks between ~4.1 and 14.3 minutes, including partially split peaks for terminally phosphorothioate‑modified fragments due to diastereomer separation.
• A total of 17 digest fragment sequences were identified (lengths 2–11 nt). These assignments were supported by accurate deconvoluted masses with mass errors within ±2.38 ppm (about half <1 ppm).
• Sequence coverage: unique fragment assignments confirmed 79% of nucleotide positions; non‑unique fragments added 16% coverage; combined coverage was 95%. A small fraction (~5%) of sequence positions was not observed in the RNase T1 digest map, with the single‑nucleotide Gp fragment particularly poorly retained and not detected.
• The resolved diastereomer peaks for phosphorothioate‑containing fragments demonstrate the column’s capability to separate closely related chemically modified oligonucleotides, which is relevant for characterizing terminal chemical protections frequently used in therapeutic guide RNAs.

Benefits and practical applications of the method

• Robust QC workflow: The combination of RNase digestion, chromatographic separation and accurate mass detection provides confident fragment identification without requiring full‑length MS/MS sequencing, which is often incomplete for long RNAs.
• Applicable to modified oligonucleotides: The method detects and resolves terminal modifications (2′‑O‑methyl, phosphorothioate) and diastereomeric species, improving characterization of therapeutic‑grade sgRNAs.
• High mass accuracy (sub‑ppm to low ppm) enables unambiguous fragment assignment and reduces false positives when matching experimental masses to expected digestion products.
• Practical throughput: straightforward sample prep (single enzyme digestion, short incubation) and relatively short LC run times facilitate routine use in development and QC labs.

Future trends and potential applications

• Parallel and orthogonal digestions: combining RNases with different specificities (or targeted RNase H with probes) to access regions not covered by RNase T1 and to increase unique sequence coverage.
• Advanced separation chemistries: greater adoption of inert‑surface oligonucleotide columns, HILIC, or mixed‑mode phases to improve retention and resolution of very small fragments (e.g., Gp) and modified species.
• Higher‑resolution and hybrid MS platforms: improved fragmentation strategies and higher MS/MS sensitivity may enable more direct full‑length sequencing of long RNAs in the future.
• Automation and informatics: automated digestion, LC/MS workflows and specialized data analysis tools will streamline sequence mapping at scale for regulatory QC of RNA therapeutics.
• Regulatory QC application: validated versions of this workflow can be implemented for batch release testing, stability studies and comparability assessments of sgRNA products.

Conclusion

The RNase T1 digestion coupled with ion‑pair reversed‑phase LC and high‑resolution Q‑TOF analysis provides a straightforward, reliable approach to sgRNA sequence mapping. For the 100‑mer sgRNA tested, intact mass confirmed full‑length material while RNase T1 fragment mapping delivered 95% sequence coverage (79% unique). The method balances practical sample preparation with chromatographic resolution and high mass accuracy, making it suitable for analytic characterization and routine quality control of modified guide RNAs.

Reference

1. Goyon A, Scott B, et al. Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–HighResolution Mass Spectrometry Analysis. Anal. Chem. 2021.
2. Gawlig C, Hirschberger R, Hanci G, et al. Full Sequencing of 100mer sgRNA via Tandem Mass Spectrometry by Targeted RNase H Digestion with Customized Probes. Anal. Bioanal. Chem. 2025. DOI: 10.1007/s00216-025-05737-y.
3. Jiang T, Yu N, Kim J, et al. Oligonucleotide Sequence Mapping of Large Therapeutic mRNAs via Parallel Ribonuclease Digestions and LCMS/MS. Anal. Chem. 2019;91(13):8500–8506. DOI: 10.1021/acs.analchem.9b01664.