RNA Analysis for CRISPR by capillary electrophoresis with laser-induced fluorescence detection

Importance of Topic

Capillary electrophoresis with laser induced fluorescence detection (CE LIF) enables simultaneous analysis of single guide RNA and Cas9 mRNA, streamlining quality control workflows for RNA based CRISPR applications. This approach addresses the need for efficient purity and integrity assessment of both small and large RNA species in gene editing and RNA therapeutic development.

Study Objectives and Overview

The study aimed to develop a single CE LIF method using one gel matrix to analyze both sgRNA and Cas9 mRNA in a single sequence run. Key goals were to achieve high resolution for short oligonucleotides, reliable size estimation for large mRNA, and reproducible quantitation of impurities and degradation products.

Methodology

RNA samples were separated using a PA 800 Plus Pharmaceutical Analysis System equipped with an LIF detector. An ssDNA 100 R gel kit provided the gel matrix. SYBR Green II dye in Tris Borate 7 M Urea buffer was used for staining. Two gel concentrations were employed: undiluted for sgRNA and pdA test mix, diluted 2.8 fold for Cas9 mRNA and large markers. Electrophoresis conditions were 30 kV for 50 minutes for short RNAs and 6 to 12.5 kV for 50 to 75 minutes for larger RNAs. Calibration was performed with an LIF performance test mix.

Used Instrumentation

• SCIEX PA 800 Plus Pharmaceutical Analysis System with 488 nm solid state laser and 520 nm emission filter
• eCAP ssDNA 100 R gel kit and DNA capillary (40.2 cm total, 30 cm effective length)
• 32 Karat software V10 for data acquisition and analysis
• SYBR Green II RNA gel stain and Tris Borate Urea buffer system

Key Results and Discussion

sgRNA analysis revealed a dominant peak at 36 minutes with multiple shorter species from 16 to 35.8 minutes representing n 1 byproducts. Corrected peak area indicated 45.07 percent purity for 100 nt sgRNA. The pdA 40 to 60 test mix demonstrated single base resolution from 15 to 60 nucleotides, detecting all expected 21 oligonucleotides and n 1 impurities. Cas9 mRNA (4.5 kb) appeared at 66.7 minutes with a main peak area of 58.08 percent; degradation fragments at 3 kb, 2.5 kb, 2 kb and 1.5 kb accounted for additional peaks. Method repeatability using RNA markers from 0.2 to 10 kb showed migration time RSD below 0.7 percent and corrected peak area RSD below 2.8 percent.

Benefits and Practical Applications

• Combined analysis of sgRNA and Cas9 mRNA in one run reduces assay time and reagent usage
• High resolution profiling enables detection of single nucleotide impurities in guide RNAs
• Size estimation of mRNA degradation products supports integrity assessment
• Robust repeatability ensures reliable quality control in RNA therapeutic manufacturing

Future Trends and Applications

Integration of on line sample preparation and capillary automation will increase throughput. Development of novel fluorescent dyes and multiplex detection schemes may allow simultaneous analysis of multiple RNA species. Enhanced software with AI driven peak identification and quantitation will streamline data interpretation. The approach can be extended to mRNA vaccines and other large RNA platforms for vaccine and therapeutic screening.

Conclusion

The described CE LIF method using a single gel matrix successfully analyzes both sgRNA and Cas9 mRNA with high resolution, accurate size estimation, and excellent repeatability. It offers a simplified, resource efficient platform for CRISPR RNA quality control, supporting pharmaceutical research and RNA therapeutic development.

References

• 1. Three ways CRISPR will change the drug world. Optum Insights, 2019.
• 2. Alt R CRISPR Cas9 sgRNAs. IDT Product Information Brochure CRS 10142 FL, 2019.
• 3. Capillary electrophoresis of oligonucleotides. Integrated DNA Technologies, 2011.