Once delivered to the cell, the crRNA and tracrRNA complex with Cas9 nuclease to generate site-specific, double-strand DNA breaks (DSBs). When DNA DSBs are repaired through non-homologous end-joining (NHEJ), the resulting small insertions and deletions (indels) can cause nonsense mutations and truncation of protein products or the introduction of a stop codon to produce gene knockouts.
UPLC (ultra-performance liquid chromatography) analysis of Edit-R tracrRNA, a >70nt purified RNA, demonstrates the high quality achieved routinely by Dharmacon 2’ACE chemical synthesis. CRISPR-Cas9 experiments using a crRNA:tracrRNA to recruit Cas9 nuclease rely on excellent purity and sequence fidelity for optimal activity. Instrument used: Acquity UPLC by Waters.