Mass Spectrometry as a Quantitative Tool for SpCas9 sgRNA Quality Control

Area of Science:

• Analytical Chemistry
• Biotechnology
• Molecular Biology

Background:

• Mass spectrometry (MS) is crucial for oligonucleotide therapeutic quality control.
• Current MS applications for single-guide RNAs (sgRNAs) are primarily qualitative.
• Accurate quantification of sgRNA variants is essential for therapeutic development.

Purpose of the Study:

• To demonstrate the quantitative capabilities of ESI-QTOF-MS for sgRNA analysis.
• To establish the limit of quantification for sgRNA length variants.
• To highlight ESI-QTOF-MS as a viable quality control method for sgRNA molecules.

Main Methods:

• Utilized electrospray-ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS).
• Analyzed a 100-nucleotide (nt) SpCas9 sgRNA.
• Determined the limits of quantification for length variants (N-4 to N+4 nucleotides).

Main Results:

• ESI-QTOF-MS assays demonstrated quantitative accuracy for sgRNA samples.
• Limits of quantification for length variants (96-104 nt) were found to be ≤1%.
• The method proved effective for analyzing chemical species within sgRNA samples.

Conclusions:

• ESI-QTOF-MS is a powerful quantitative tool for sgRNA analysis.
• This technique significantly enhances the quality control of oligonucleotide therapeutics.
• The study validates ESI-QTOF-MS for precise sgRNA characterization.