Unveiling the Structural Complexity of Guide RNA, A Critical Reagent Used in CRISPR Gene Therapy

The guide RNA (gRNA), a pivotal component in the CRISPR/Cas9 genome editing system, confers its target specificity. gRNAs are often manufactured using solid-phase synthesis methods, which can lead to complex impurity profile and higher order structures. The analysis of gRNA is rendered challenging due to its large size, intricate impurity profile, and complex secondary structure. In response to these challenges, we developed several liquid chromatography-based platform methods, such as ion pairing reversed phase liquid chromatography (IP-RPLC), hydrophilic interaction liquid chromatography (HILIC), ion exchange chromatography (IEC), and size exclusion chromatography (SEC) for comprehensive gRNA analysis. Furthermore, we revealed the change of higher order structure of gRNA with a combination of temperature dependent SEC and microfluidic modulation spectroscopy (MMS) method.