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Top-Down Mass Spectrometry of Synthetic Single Guide Ribonucleic Acids Enabled by Facile Sample Clean-Up
Christopher M Crittenden1, Michael B Lanzillotti2, Bifan Chen1
1Small Molecule Analytical Chemistry, Genentech Inc., South San Francisco, California 94080, United States.
Analytical Chemistry
|January 6, 2023
Summary
We developed a new mass spectrometry method to easily sequence large single guide RNA molecules used in CRISPR gene editing. This technique improves accuracy for essential biomedical research and therapeutic development.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- CRISPR-Cas9 genome editing relies on single guide ribonucleic acids (sgRNAs) for sequence-specific DNA targeting.
- sgRNA design and quality are critical for efficient and specific genome editing outcomes.
- Traditional mass spectrometry (MS) faces challenges in analyzing large oligonucleotides due to salt and adduct interference.
Purpose of the Study:
- To develop an improved mass spectrometry workflow for characterizing and sequencing large sgRNAs.
- To overcome limitations in desalting and achieving high-quality spectra for intact oligonucleotides.
- To enable facile top-down analysis of chemically modified oligonucleotides without extensive sample preparation.
Main Methods:
- Development of an online column-based clean-up method using reversed-phase chromatography in size exclusion mode.
- Application of a top-down mass spectrometry approach without nuclease digestion.
- Utilizing higher-energy collision dissociation (HCD), collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and activated electron photodetachment (a-EPD) for fragmentation.
Main Results:
- The online clean-up method effectively removed salts and metal adducts from large oligonucleotides.
- A 100-nucleotide sgRNA was sequenced using liquid chromatography-tandem MS (LC-MS/MS) in a 10-minute run.
- CID provided 67% sequence coverage, while combined CID, UVPD, and a-EPD achieved 80% overall coverage and 100% cleavage for the variable 5' region.
Conclusions:
- The developed LC-MS/MS platform offers a simple and effective top-down workflow for analyzing and sequencing large oligonucleotides.
- This method facilitates the characterization of chemically modified sgRNAs, crucial for CRISPR technology advancement.
- The approach eliminates the need for extensive sample treatment, streamlining oligonucleotide analysis.
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