Development of an ion pairing reversed-phase liquid chromatography-mass spectrometry method for characterization of clustered regularly interspaced short palindromic repeats guide ribonucleic acid

Guide ribonucleic acid (gRNA) is a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. The single stranded guide RNA (sgRNA) is the most commonly used gRNA in application. Evaluation of the impurity profile of synthetic sgRNA is important for any CRISPR genome editing experiments. However, the large molecular size, complex impurity profile and unique secondary structure pose many challenges in the analysis of sgRNA by ion pairing reversed-phase liquid chromatography (IP-RPLC), the commonly used method. In this work, we developed a generic IP-RPLC method for guide RNA analysis. We found that large pore size of stationary phase was the most critical column parameter to achieve high resolution separation of sgRNA while particle structure, particle size and surface chemistry had less impact. Our results indicated that charge interaction was the most critical mechanism for retention and mass transfer had less impact on the performance of separation. An IP-RPLC/mass spectrometry (MS) method was also developed with a specific practice to reduce adducts and enable intact MS analysis of sgRNAs. The generic IP-RPLC method demonstrates its feasibility to serve as a release, stability, characterization and in-process control testing method for synthetic sgRNA products.