High Resolution Ion Mobility Mass Spectrometry Enables Sequencing and Characterization of Synthetic Guide RNA

Single guide RNA (sgRNA) enables CRISPR-based editing and is a critical component of the development of Cell Therapies. These synthetic RNAs are approximately 100 nucleotides long and are often heavily modified to enhance stability and prevent premature degradation by nucleases. In addition, they posses significant secondary structure that is critical for their function. In order to ensure efficacy and patient safety, sgRNAs used in cell therapies need to be well characterized. Verification of the primary structure (sequence) of the guide RNA is highly important and required by regulatory agencies. Several methods have been developed that use mass spectrometry to verify the sgRNA sequence. This presentation will show how we have used traveling wave ion mobility spectrometry (TWMS) using structures for lossless ion manipulation (SLIM) to enable rapid sequencing of sgRNA when coupled with high resolution mass spectrometry. The developed workflow enables the sequencing of sgRNA and the characterizations of some of the RNA-modifications present in the sequence.