Quantitative Analysis of Phosphorothioate Isomers in CRISPR sgRNA at Single-Residue Resolution Using Endonuclease Digestion Coupled with Liquid Chromatography Cyclic Ion Mobility Mass Spectrometry (LC/cIMS).

ABSTRACT: Phosphorothioate (PS) modifications in single-guided RNA (sgRNA) are crucial for genome editing applications using the CRISPR/Cas9 system. These modifications may enhance sgRNA stability, pharmacokinetics, and binding to targets, thereby facilitating the desired genetic alterations. Incorporating multiple PS groups at varying positions may introduce chiral centers into the sgRNA backbone, resulting in a complex mixture of constitutional- and stereoisomers that challenges current analytical capabilities for reliable identification and quantification. In this study, we developed an innovative methodology that combines endonuclease digestion of sgRNA with ion pairing reversed-phase liquid chromatography coupled with cyclic ion mobility mass spectrometry (IPRP-LC/cIMS) to fully distinguish PS-

SUBMITTER: Su Y 

PROVIDER: S-EPMC11800180 | biostudies-literature | 2025 Feb

REPOSITORIES: biostudies-literature