The advancement of CRISPR-based gene editing tools into biotherapeutics offers the potential for cures to genetic disorders and for new treatment paradigms for even common diseases. Arguably, the most important component of a CRISPR-based medicine is the guide RNA, which is generally large (>100-mer) synthetic RNA composed of a "tracr" and "spacer" region, the latter of which dictates the on-target editing site as well as potential undesired off-target edits. Aiming to advance contemporary capabilities for gRNA characterization to ensure the spacer region is of high fidelity, top-down mass spectrometry was herein implemented to provide direct and quantitative assessments of highly modified gRNA. In addition to sequencing the spacer region and pinpointing modifications, top-down mass spectra were utilized to quantify single-base spacer substitution impurities down to <1% and to decipher highly dissimilar spacers. To accomplish these results in an automated fashion, we devised custom software capable of sequencing and quantifying impurities in gRNA spacers. Notably, we developed automated tools that enabled the quantification of single-base substitutions, including advanced isotopic pattern matching for C > U and U > C substitutions, and created a de novo sequencing strategy to facilitate the identification and quantification of gRNA impurities with highly dissimilar spacer regions.
© 2023 The Authors. Published by American Chemical Society.