Agilent 6530b quadrupole time of flight mass spectrometry

The prime editing guide RNA (pegRNA) was synthesized using Synthego’s CRISPRevolution platform with solid-phase phosphoramidite chemistry. Based on the original prime editing report [24 (link)], we selected a reverse transcriptase (RT) template of 10 nucleotides in length, inclusive of the C>G transversion, and a primer binding site (PBS) of 16 nucleotides in length. Three 2′-O-methyluridinylates were attached at the 3′ end of the PBS and stabilized with phosphorothioate backbones. The first three bases of the protospacer were modified as 2′-O-methyl derivatives and stabilized as phosphorothioates. The pegRNA was purified using reversed-phase high-performance liquid chromatography (Buffer A, 0.1 M TEAA; Buffer B, 50% 0.1 M TEAA/50% acetonitrile, 15%–95% B gradient in 15 min), and their identities were confirmed using an Agilent 1290 Infinity II liquid chromatography system coupled with Agilent 6530B Quadrupole time-of-flight mass spectrometry (Agilent Technologies, Santa Clara, CA) in a negative ion polarity mode.