Spacer Fidelity Assessments of Guide RNA by Top-Down Mass Spectrometry
ABSTRACT: 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 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.
A list of most relevant published research articles where Mike Freitas, CSO, has led and/or participated in as researcher and/or author.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759086/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749473/
https://academic.oup.com/bioinformatics/article/25/10/1341/270609
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8431783/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775432/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749500/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4141472/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405592/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2614441/
https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-8-133
https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-11-436
https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-9-347