Quality assessment of sgRNA and Cas9 mRNA for CRISPR – based therapeutics by capillary electrophoresis (CE)

Gene editing is one of the fastest-growing areas in biotechnology and biopharmaceutical research, with CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) leading the field as the primary method.

A common strategy involves co-delivering single-guide RNA (sgRNA) and Cas9 mRNA to enhance genome editing efficiency. High-resolution characterisation of both sgRNA and Cas9 mRNA in a single analysis for CRISPR/Cas9 applications was previously demonstrated.

This technical note highlights the assessment of sgRNA quality with single-nucleotide resolution, as well as the accurate ratio of sgRNA and Cas9 mRNA for CRISPR-based therapeutics through direct measurements with UV detection.

Introduction

CRISPR is an immunological defense mechanism used by bacteria to combat invading pathogens. Cas9 is an RNA-guided DNA endonuclease. Due to its ability to perform site-directed DNA cleavage and trigger homologous recombination, Cas9 has been utilised as a gene-editing tool to introduce gene inactivation and genome modification. In the pharmaceutical industry, CRISPR can enhance existing therapies, aid in identifying drug targets, and facilitate the testing of drug candidates.

Two RNA molecules are essential for CRISPR: sgRNA and Cas9 mRNA. Purity analysis of sgRNA and Cas9 mRNA is a critical quality attribute for CRISPR-Cas9-based therapeutics. Accurate purity assessment of sgRNA requires single-nucleotide resolution to separate n-1 species from the full-length sgRNA.

However, common analytical methods, such as liquid chromatography or denaturing agarose gels, have challenges in resolving n-1 species from full-length, long oligos, such as sgRNAs. Additionally, during the development of CRISPR Cas9-based RNA therapeutics, it is often advantageous to co-deliver the sgRNA and the Cas9 mRNA simultaneously.

Key features

High-resolution separation of n-1 from full-length sgRNA: Enables dose-dependent detection of low levels of n-1 species and accurate purity assessment of sgRNA.
Excellent assay repeatability in detecting low levels of n-1 spiked in full-length sgRNA: Ensures consistent and reliable analysis for the n-1 species, demonstrating high repeatability with low %RSD in CPA%.
Accurate ratio determination of sgRNA and Cas9 mRNA: Provides direct measurements of the amounts of sgRNA and Cas9 mRNA by UV detection at 254 nm, demonstrating high correlation between the theoretical and measured ratio of sgRNA to Cas9 mRNA.
Streamlined operation with ready-to-use, intuitive kits: Ensures a simple, efficient workflow with consistent results.