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.
R1006 encodes Streptococcus pyogenes Cas9 and is used with guide RNA for sequence-directed DNA cleavage in genome editing.
2. Which cap and modified nucleotides does R1006 use?
R1006 is ARCA capped, poly(A)-tailed, and contains 5mCTP and ψUTP. Consult the current batch documentation for product-specific specifications.
3. How does R1006 differ from the EZ Cap™ Cas9 variants?
R1006 is the ARCA-capped, 5mCTP/ψUTP variant. R1014 and R1015 are separate EZ Cap™ Cas9 products with m1Ψ and 5-moUTP modifications, respectively.
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.
