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.

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A list of most relevant published research articles where Mike Freitas, CSO, has led and/or participated in as researcher and/or author.

Xu, Hua, and Michael A Freitas. “MassMatrix: a database search program for rapid characterization of proteins and peptides from tandem mass spectrometry data.” Proteomics vol. 9,6 (2009): 1548-55. doi:10.1002/pmic.200700322
Xu H, Zhang L, Freitas MA. Identification and characterization of disulfide bonds in proteins and peptides from tandem MS data by use of the MassMatrix MS/MS search engine. J Proteome Res. 2008 Jan;7(1):138-44. doi: 10.1021/pr070363z. Epub 2007 Dec 12. PMID: 18072732; PMCID: PMC2749473.
Hua Xu, Michael A. Freitas, Automated diagnosis of LC-MS/MS performance, Bioinformatics, Volume 25, Issue 10, May 2009, Pages 1341–1343,
Gardner ML, Freitas MA. Multiple Imputation Approaches Applied to the Missing Value Problem in Bottom-Up Proteomics. Int J Mol Sci. 2021 Sep 6;22(17):9650. doi: 10.3390/ijms22179650. PMID: 34502557; PMCID: PMC8431783.
Xu H, Wang L, Sallans L, Freitas MA. A hierarchical MS2/MS3 database search algorithm for automated analysis of phosphopeptide tandem mass spectra. Proteomics. 2009 Apr;9(7):1763-70. doi: 10.1002/pmic.200800282. PMID: 19288523; PMCID: PMC2775432.
Xu H, Freitas MA. Monte carlo simulation-based algorithms for analysis of shotgun proteomic data. J Proteome Res. 2008 Jul;7(7):2605-15. doi: 10.1021/pr800002u. Epub 2008 Jun 11. PMID: 18543962; PMCID: PMC2749500.
Xu H, Hsu PH, Zhang L, Tsai MD, Freitas MA. Database search algorithm for identification of intact cross-links in proteins and peptides using tandem mass spectrometry. J Proteome Res. 2010 Jul 2;9(7):3384-93. doi: 10.1021/pr100369y. PMID: 20469931; PMCID: PMC4141472.
Zhang L, Gardner ML, Jayasinghe L, Jordan M, Aldana J, Burns N, Freitas MA, Guo P. Detection of single peptide with only one amino acid modification via electronic fingerprinting using reengineered durable channel of Phi29 DNA packaging motor. Biomaterials. 2021 Sep;276:121022. doi: 10.1016/j.biomaterials.2021.121022. Epub 2021 Jul 10. PMID: 34298441; PMCID: PMC8405592.
Zhang L, Xu H, Chen CL, Green-Church KB, Freitas MA, Chen YR. Mass spectrometry profiles superoxide-induced intramolecular disulfide in the FMN-binding subunit of mitochondrial Complex I. J Am Soc Mass Spectrom. 2008 Dec;19(12):1875-86. doi: 10.1016/j.jasms.2008.08.004. Epub 2008 Aug 12. PMID: 18789718; PMCID: PMC2614441.
Xu, H., Freitas, M.A. A mass accuracy sensitive probability based scoring algorithm for database searching of tandem mass spectrometry data. BMC Bioinformatics 8, 133 (2007).
Xu, H., Freitas, M.A. A Dynamic Noise Level Algorithm for Spectral Screening of Peptide MS/MS Spectra. BMC Bioinformatics 11, 436 (2010).
Xu, H., Yang, L. & Freitas, M.A. A robust linear regression based algorithm for automated evaluation of peptide identifications from shotgun proteomics by use of reversed-phase liquid chromatography retention time. BMC Bioinformatics 9, 347 (2008).