The CRISPR/Cas9 system is increasingly used as a genome editing tool with applications that include disease treatment. The Cas9 endonuclease can cleave off-target sites when the single guide RNA (sgRNA) recognizes genome loci that are similar to the target DNA, resulting in disruptions to the functionality and stability of normal genes. To overcome the frequent off-target cleavage by the wild-type Cas9 endonuclease, engineered Cas9 variants with amino acid mutations have been developed. On the other hand, PTMs that can modify protein structure and hydrophobicity also have an important impact on protein function and stability. Thus, a comprehensive characterization of engineered Cas9 that can confirm the amino acid sequence and PTMs is important to ensure its efficacy.

This Masterclass LIVE demonstrates an LC-MS/MS approach for the comprehensive characterization of an engineered Cas9 protein that achieved <95% sequence coverage and confident identification of PTMs.

The CRISPR/Cas9 system relies on quality reagents to ensure high editing efficiency. Chemically synthesized guide RNA can include stabilizing modifications, increasing sequence lengths, and secondary structure which can result in final product heterogeneity. Additionally, Cas9 mRNA can be used to deliver Cas9 protein in RNA-based gene editing systems. A workflow capable of analyzing a wide RNA size range can enable simultaneous purity analysis of guide RNA and Cas9 mRNA, streamlining purity analysis for complete CRISPR gene editing systems.

This Masterclass LIVE shows you how you can ensure accurate purity value determination of guide RNA structures and analyze long length IVT mRNA sequences.