Analytical Assessment of sgRNA Impurities and Their Impact on Functional Performance

Single guide RNA (sgRNA) is a critical component of the clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system, guiding Cas9 to specific genomic loci for precise DNA modification. With its growing clinical potential, sgRNAs have emerged as a promising modality for gene editing-based therapeutics, underscoring the need for robust analytical characterization to ensure quality, safety, efficacy, and regulatory compliance. Here, we present an integrated strategy for deep profiling of sgRNA impurities, combining high-resolution ion-pairing reversed-phase liquid chromatography (IP-RPLC) and size-exclusion chromatography (SEC) with advanced technologies, namely, native mass spectrometry and nanopore direct RNA sequencing. The isolation and characterization of isolated chromatographic peaks revealed truncated, chemically modified, and deletion-prone species. Notably, early eluting fractions in IP-RPLC exhibited elevated deletion frequencies in the target-specific region, correlating with reduced gene-editing efficiency and increased variability in T cells when tested using the Cas-CLOVER system. In contrast, late-eluting fractions in IP-RPLC revealed polyphosphorylated variants with minimal functional impact, while off-target analyses suggested that early eluting impurities may paradoxically reduce off-target editing. Lastly, even at high levels, sgRNA aggregates showed only a limited impact on activity: fully aggregated preparations displayed ∼10% lower knockout efficiency; however, an increase in cumulative off-target frequencies was observed. Overall, this study highlights the value of combining advanced analytical tools to achieve deep profiling of sgRNA impurities. By linking specific impurity profiles to functional outcomes, these findings provide actionable insights for improving sgRNA quality control and advancing the development of safe and effective gene editing-based therapeutics.