CRISPR Single Guide RNA Characterization by IP- RP-LC-MS with a Premier Oligonucleotide BEH 300 Å C18 Column

Importance of the topic

CRISPR/Cas genome editing relies on the precision and integrity of a single guide RNA (sgRNA). Reliable characterization of sgRNA ensures accurate targeting, minimizes off-target effects, and supports therapeutic, diagnostic, and research applications.

Study objectives and overview

This work evaluates ion-pairing reversed-phase liquid chromatography coupled with mass spectrometry (IP-RP-LC-MS) using a Premier Oligonucleotide BEH 300 Å C18 column for comprehensive analysis of intact and RNase T1-digested sgRNA. The goal is to demonstrate high resolution separations, intact mass determination, and detailed oligo mapping to verify sequence integrity and chemical modifications.

Methodology and instrumentation

Two analytical workflows were applied:

• Intact sgRNA analysis by IP-RP-LC-MS for direct mass measurement and purity assessment.
• RNase T1 digestion followed by oligo mapping and MSE fragmentation for sequencing and modification localization.

Instrumentation used

• Waters ACQUITY UPLC I-Class FTN system with Tunable UV detector (260/280 nm)
• Acquity Premier Oligonucleotide BEH C18 WidePore column (300 Å, 1.7 µm, 2.1×100/150 mm)
• BioAccord LC-MS System with RDa high-resolution detector
• Waters Vion QTof mass spectrometer (30 K resolution)
• RNase T1 enzyme for controlled digestion
• waters_connect platform integrating Intact Mass, UNIFI Scientific Library, and CONFIRM Sequence applications

Main results and discussion

Intact analysis resolved failure sequences and degradants, yielding a deconvoluted mass of 32,242 Da (vs. expected 32,240 Da). RNase T1 digestion produced unique oligonucleotide fragments covering 82 % of the sequence (98 % including non-unique fragments). High chromatographic resolution separated intact and partial-thiolated termini diastereomers. MSE fragmentation coupled with in silico digestion libraries enabled confident assignment of sequence-informative ions and detected incomplete phosphorothioate modifications at both 5′ and 3′ ends.

Benefits and practical applications of the method

• High-speed, robust assessment of sgRNA purity and integrity
• Direct intact mass confirmation without cDNA conversion
• Detailed oligo mapping for sequence verification and modification mapping
• Detection of synthesis-related heterogeneity (phosphorothioate diastereomers, incomplete thiolation)
• Seamless integration with application-based software for data processing and reporting

Future trends and opportunities

Ongoing developments may include broader nuclease panels for complete sequence coverage, alternative column chemistries for enhanced resolution of modified oligonucleotides, advanced informatics for automated variant detection, and application of this workflow to novel CRISPR systems and therapeutic manufacturing quality control.

Conclusion

The IP-RP-LC-MS workflow using a Premier Oligonucleotide BEH C18 column provides a comprehensive, high-resolution approach for both intact and digested sgRNA characterization. It delivers accurate mass determination, detailed sequence mapping, and reliable detection of chemical modifications critical for ensuring CRISPR reagent quality.

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