(W1130-06-39) Bioanalysis of Endonuclease Cas9 in Plasma Conducing to Clarify CRISPR-Cas9-Mediated Genome Engineering Using Immunocapture followed with LC-MS/MS Screener Application

Purpose: During the last several years, the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing technology has swept the scientific community, heralding a novel era of disease detection and molecular therapeutics. Cas9, a CRISPR-associated endonuclease, can be targeted to specific genomic loci by single guide RNAs (sgRNAs) capable of site-specific double-strand cleavage in DNA, which has been widely used to efficiently modify endogenous genes in a wide variety of biologically functional cell types and in organisms that have traditionally been challenging to work with genetically. To clarify the CRISPR-Cas9-mediated genome engineering, the bioanalysis of streptococcus pyogene Cas9 protein in rat and monkey plasma was developed and reported here.

Methods: Magnetic bead-based immunocapture followed with liquid-chromatography coupled with mass spectrometry (LC–MS/MS), not ligand-binding assay (LBA), was used due to its adjustable LC separation capability for isolating interferences and high specificity of the MS nature.

Results: Firstly, on the basis of immunocapture against N-terminal, signature peptide YFDTTIDR near C-terminal and TILDFLK located at the middle of the Cas9 sequence were selected and used for qualitative analysis and quantitative analysis of Cas9 respectively, ensuring both the analysis of complete protein molecule and good sensitivity. Secondly, establishing a fast and stable LC-MS method for the bioanalysis of Cas9 protein without any interferences was also achieved. As a result, the quantification of Cas9 protein based on bottom-up approach was realized, in which up to 2000 times curve range and high sensitivity (LLOQ=5.0 ng/mL) was acquired with optimal reproducibility.

Conclusion: Using the optimized method, a sensitive and reproducible LC-MS/MS assay was successfully developed to quantify Cas9 in rat and monkey plasma. The pharmacokinetics and toxicokinetics data acquired using the newly-developed bioanalytical method for the Cas9 protein would provide insights to the study of mechanisms and metabolism of the gene editing tool in vivo.

References: 1. Javaid D, Ganie S Y, Hajam Y A, et al. CRISPR/Cas9 system: a reliable and facile genome editing tool in modern biology [J]. Molecular Biology Reports, 2022. 49: 12133-12150.
2. Sternberg S H, Redding S, Jinek M, et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9[J]. Nature, 2014. 507: 62-66.

CRISPR-Cas9-mediated Genome Engineering Method

The Workflow for Bioanalysis of Cas9 Protein

Th Standard Curve of Cas9 Protein in Monkey Plasma