Mass Spectrometry as a Quantitative Tool for SpCas9 sgRNA Quality Control.

Mass spectrometry (MS) has long been used for quality control of oligonucleotide therapeutics, including single-guide RNAs (sgRNAs) for clustered regularly interspaced short palindromic repeats techniques. However, the application of MS is limited to qualitative assays in most cases. Here, we showed that electrospray-ionization quadrupole time-of-flight MS (ESI-QTOF-MS) assays can be quantitative for chemical species found in sgRNA samples. More specifically, using a 100-nt SpCas9 sgRNA as the example, we estimated that the limits of quantification for length variants in the range of N - 4 to N + 4 (i.e., 96-104 nucleotides) were equal to or lower than 1%. Our study highlighted the potential of ESI-QTOF in its application as a quality control method for sgRNA molecules.