Assessing mRNA and sgRNA Quality for Cell and Gene Therapy Applications Using Nanopore Direct RNA Sequencing

Recent advances in RNA technology have enabled the development of diverse therapeutics spanning vaccines, immunotherapies, and genome-editing platforms. Ensuring clinical efficacy and safety requires precise characterization and control of RNA critical quality attributes (CQAs). Nanopore direct RNA sequencing (NDRS) has emerged as a powerful single-molecule analytical approach capable of simultaneously resolving sequence and structural features consistent with regulatory expectations. In this study, we establish NDRS as a comprehensive, multiattribute analytical platform by integrating novel strategies to assess key CQAs in a single assay. Following workflow optimization, NDRS accurately determined full-length mRNA sequences and evaluated transcript integrity. Notably, we developed the first sequencing-based method for quantifying 5' capping efficiency directly from native RNA molecules. Additionally, we demonstrated, for the first time, full-length sequencing of 100-nucleotide single-guide RNA (sgRNA) molecules by incorporating a 5' RNA oligo adapter, enabling complete identity verification. Quantitative results for poly(A) tail length, integrity, and capping efficiency were consistent with established orthogonal techniques, including chromatography and mass spectrometry. Moreover, functional correlation studies with Cas9 mRNA and sgRNA used in CRISPR-Cas9 editing revealed that increased mRNA degradation led to decreased knockout efficiency. Together, these findings position NDRS as a versatile and unified analytical platform for comprehensive characterization of mRNA and sgRNA, supporting quality assurance, comparability, and control in the development and manufacturing of next-generation RNA therapeutics.