Analysis of crRNA Using Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC ESI MS)

Mass spectrometry is a powerful tool for characterizing RNA. Here we describe a method for the identification and characterisation of crRNA using liquid chromatography interfaced with electrospray ionization mass spectrometry (LC ESI MS). The direct purification of crRNA from the Cascade-crRNA complex was performed using denaturing ion pair reverse phase chromatography. Following purification of the crRNA, the intact mass was determined by LC ESI MS. Using this approach, a significant reduction in metal ion adduct formation of the crRNA was observed. In addition, RNase mapping of the crRNA was performed using RNase digestion in conjunction with liquid chromatography tandem MS analysis. Using the intact mass of the crRNA, in conjunction with RNase mapping experiments enabled the identification and characterisation of the crRNA, providing further insight into crRNA processing in a number of type I CRISPR-Cas systems.