SpCas9 mRNA expresses an optimized version of Streptococcus pyogenes protein. It can be used with a purified guideRNA sequence for genomic DNA target recognition and cleavage in the CRISPR-Cas genome editing system.
Type II CRISPR-Cas system from the bacterium S. pyogenes has been adapted for genome engineering in eukaryotic systems, which requires two main components: the endoculease Cas9 and non-coding guide RNA (gRNA). The Cas9 protein can be programmed with gRNA, targeting a site-specific DNA double-strand break (DSB).
Our SpCas9 mRNA is provided at a concentration of 1mg/ml, it is ARCA capped, polyA-tailed and incorporated with modified nucleotides. The capping of ARCA (anti-reverse cap analog) ensures high translation efficacy, while the addition of 5mCTP/ψUTP and poly(A) tail reduces host cell immune response and enhance mRNA stability.
Moreover, Cas9 mRNA expresses the Cas9 protein with two nuclear localization signal (NLS) and C terminal HA epitope tag that enhance the rate of DNA cleavage and facilitate downstream purification.
1. How do I choose the right RNA product for my research?
It can be based on your experimental goals:
For Tracking Transfection and Translation Efficiency: APExBIO Reporter Gene mRNAs (e.g. EGFP, Firefly Luciferase mRNA) are commonly used to track transfection efficiency and protein expression duration; evaluate gene expression and cell viability; study mRNA localization and bio-distribution via in vivo imaging; optimize transfection conditions and validate LNP delivery system.
For Gene Editing, Functional Studies and Gene Therapy Research: APExBIO offers various functional protein mRNAs, involving tumor suppressors (e.g. p53, PTEN), cytokines (e.g. IL-12, IL-10), gene-editing tools (e.g. spCas9, Cre Recombinase), gene replacement protein (e.g. EPO), and antigens (e.g. OVA, SARS-CoV-2 Spike).
For Sustained Protein Expression: APExBIO Self-amplifying RNA (saRNA) and Circular RNA (circRNA) are recommended for applications requiring prolonged protein expression. saRNA enables lasting and strong protein expression at lower doses, while circRNA has enhanced structural stability and extended expression duration.
2. What are the key advantages of APExBIO mRNA products?
Advanced Capping Technology: Utilizes Cap 1 structure (EZ Cap™ Cap) to achieve enhanced translation efficiency and minimizing activation of the host innate immune response. The capping efficiency can reach 90–99%.
Diverse Modification Options: Provides a range of modified nucleotides, such as m1Ψ B8049, 5-moUTP B8061 and Cy5-UTP B8333, which reduce immunogenicity, improve mRNA stability, and maximize protein expression levels.
Stringent Quality Control: Each batch undergoes rigorous quality assessment including capping efficiency, purity, integrity, and sterility to ensure batch-to-batch consistency.
3. Why is Capping technology important, and how does it affect mRNA stability and immunogenicity?
The 5′ Cap structure protects mRNA from degradation and is required for ribosome recognition and translation initiation.
Cap 1, as a next-generation cap analog, closely mimics the natural structure of endogenous eukaryotic mRNA and is therefore recognized by cells as “self RNA.” This effectively reduces innate immune activation and enables efficient protein expression.
All APExBIO EZ Cap™ mRNA products utilize Cap 1 technology.
4.What other factors are critical in mRNA synthesis?
In addition to 5’ Cap, a complete in vitro transcribed (IVT) mRNA includes:
• Optimized 5′ and 3′ untranslated regions (UTRs) to enhance stability and translation.
• A Poly(A) tail (typically ~100 nucleotides) protects mRNA and facilitates translation.
• Suitable nucleotide modifications based on the downstream applications.
• Codon optimization based on the host expression system.
5. How to choose mRNA modifications based the downstream applications?
m1Ψ (N1-methyl-pseudouridine): Strongly reduces innate immune responses and enhances protein expression. Widely used in mRNA vaccines research.
ψUTP (pseudouridine), 5-moUTP (5-methoxyuridine), and 5mCTP (5-methylcytidine): Reduce immunogenicity while improving mRNA stability and translational efficiency. 5mCTP is commonly used in saRNA.
Fluorescent- or Biotin-labeled Nucleotides (e.g. Cy5-UTP, Biotin-UTP): Used for visualization of mRNA to track mRNA delivery and localization.
6. What are the precautions for handling RNA?
RNA is highly susceptible to degradation.
Thawing: Before use, allow the product to thaw completely on ice.
Aliquoting: Briefly centrifuge and aliquot upon first use to avoid repeated freeze–thaw cycles.
RNase Contamination Control: Use RNase-free consumables and reagents; maintain a clean and RNase-free working environment.
Transfection: Always mix mRNA with transfection reagent first before adding to serum-containing medium to prevent RNase degradation.
7. What should I do if protein expression is lower than expected?
Check RNA Integrity: Make sure the RNA has been properly stored and handled according to the instruction, and avoid repeated freeze–thaw cycles.
Check Transfection Procedure: Use reporter mRNA (such as EZ Cap™ Firefly Luciferase mRNA) as a positive control to evaluate transfection reagents, cell conditions and lab protocols.
Optimize Experiments: Consider adjusting the RNA-to-transfection reagent ratio, cell density, incubation time, and protein detection time points etc.
If the issue can’t be resolved, please contact our technical support team with detailed data for further assistance.
8. What if there are issues during shipment?
All RNA products are shipped on dry ice to maintain product integrity. If the dry ice has melted (and the product feels warm) or if the package is damaged upon receipt, please take photos immediately and contact us as soon as possible.
