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Overview
This GenCefe mRNA encodes SpCas9, a cas protein construct supplied for gene editing and genome engineering applications. The product is formulated as lyophilised, non-encapsulated RNA and is intended for use in cell-based research applications requiring transient protein expression with reduced immunogenicity.
mRNA Construct Design
- 5′ Cap: Cap1 (m7GpppNm) — co-transcriptionally added during in vitro transcription (IVT). Cap1 includes 2′-O-methylation at the first transcribed nucleotide, closely mimicking the cap structure found on endogenous mammalian mRNA and reducing recognition by innate immune sensors (e.g., IFIT1/IFIT3).
- Modified Nucleotides: 100% N1-methylpseudouridine (m1Ψ; N1-Me-Pseudo UTP) substitution for all uridine residues. m1Ψ modification reduces TLR7/TLR8-mediated innate immune activation and PKR-driven translational suppression, resulting in improved protein expression in immunocompetent cells and primary cell types.
- Poly(A) Tail: 100–120 nt — enzymatically polyadenylated. The poly(A) tail stabilises the 3′ terminus, supports poly(A)-binding protein (PABP) recruitment, and enhances ribosome recycling for efficient cap-dependent translation.
- 5′ UTR: hHBA1 (hemoglobin subunit alpha 1 5′ UTR) — a well-characterised human UTR that supports efficient cap-dependent translation initiation.
- 3′ UTR: hHBA1 (hemoglobin subunit alpha 1 3′ UTR) — provides post-transcriptional stability and modulates mRNA decay kinetics.
- Signal Peptide: No
- Protein Tag: No
- Codon Optimisation: No (native human codon usage retained)
- mRNA Length: Provided upon order placement.
- Form: Lyophilised powder; reconstitute in DEPC-treated water as needed.
This mRNA is supplied as non-encapsulated, lyophilised powder. Delivery vehicle selection (LNP, electroporation, lipofection) is at the discretion of the end user and should be optimised for the target cell type and application.
Biological Background
CRISPR-associated (Cas) proteins are programmable RNA-guided endonucleases derived from bacterial adaptive immune systems. Class 2 Cas nucleases, including the widely used Streptococcus pyogenes Cas9 (SpCas9) and the Cpf1 (Cas12a) orthologues from Acidaminococcus (AsCas12a/AsCpf1) and Lachnospiraceae (LbCas12a/LbCpf1), introduce site-specific double-strand breaks guided by single guide RNA (sgRNA) or CRISPR RNA (crRNA). Delivery of Cas nuclease as mRNA rather than plasmid DNA reduces the risk of off-target genomic integration, restricts editing activity to the transient window of mRNA half-life, and minimises innate immune responses in primary cells, making mRNA-based CRISPR delivery increasingly preferred for therapeutic genome editing and ex vivo haematopoietic stem cell (HSC) modification.
Research Relevance and Current Trends
- Non-integrating genome editing: Cas9 and Cas12a mRNA co-delivery with guide RNA (as synthetic crRNA:tracrRNA duplexes or sgRNA) achieves transient editing activity, reducing the off-target editing window compared to plasmid-based systems.
- Base editing and prime editing: Modified Cas protein mRNAs (e.g., high-fidelity SpCas9 variants) serve as the nuclease backbone for base editors (CBEs/ABEs) and prime editors, where mRNA delivery reduces immunogenicity and enables editing in post-mitotic cells.
- Clinical-grade HSC editing: Ex vivo haematopoietic stem cell editing programmes (e.g., for haemoglobinopathies) favour mRNA-RNP delivery for regulatory safety profiles in GMP-adjacent workflows.
Common Research Applications
- Transient genome editing — Cas9/Cas12a mRNA co-delivery with guide RNA for site-directed insertions, deletions, or substitutions in primary cells and cell lines.
- Indel analysis — mRNA-based editing followed by surveyor assay, Sanger sequencing, or NGS to quantify on-target editing efficiency at defined genomic loci.
- Base editing and prime editing — Cas mRNA as the scaffold for base editor or prime editor fusion proteins for precise nucleotide conversions without double-strand breaks.
- RNP optimisation — Cas protein mRNA-to-guide-RNA ratio titration to maximise editing efficiency while minimising cytotoxicity across cell types.
Notes for Experimental Interpretation
- Cas protein mRNA activity depends on guide RNA quality and design; evaluate multiple guide sequences across the target locus and confirm on-target editing by sequencing before committing to functional experiments.
- High Cas9 mRNA doses can be cytotoxic, particularly in primary cells; titrate mRNA amount empirically and assess viability 24 h post-transfection before proceeding to editing analysis.
- eSpCas9 (enhanced specificity variant) contains specific mutations (e.g., K848A/K1003A/R1060A) that reduce off-target activity; confirm the variant designation matches the intended specificity profile.
- Cas12a (Cpf1) orthologs have distinct PAM requirements (TTTV for LbCas12a; TTTV/TTTT for AsCas12a); verify PAM compatibility at the target site before designing crRNA.
Synthetic mRNA products typically incorporate chemical modifications to minimize innate immune recognition. The most widely used modification is N1-methylpseudouridine (m1Ψ) substitution at all uridine positions, which reduces activation of Toll-like receptors (TLR7/TLR8) and protein kinase R (PKR), resulting in improved translational efficiency and a reduced inflammatory response. The 5′ cap structure is equally important: Cap1 (m7GpppNm), which includes 2′-O-methylation at the first transcribed nucleotide, closely mimics endogenous mammalian mRNA and limits recognition by innate immune sensors such as IFIT1 and IFIT3. Together, these modifications support more robust and sustained protein expression in research applications.
Synthetic mRNA is highly sensitive to ribonuclease (RNase) degradation and must be handled carefully. Lyophilized products should be stored at −20°C; aqueous formulations should be kept at −70°C or below. Repeated freeze-thaw cycles should be avoided — aliquoting immediately upon receipt is strongly recommended. All handling must be performed in an RNase-free environment using dedicated pipettes, nuclease-free consumables, and DEPC-treated or certified nuclease-free water. RNA integrity should be confirmed by agarose gel electrophoresis or capillary electrophoresis (e.g., Bioanalyzer or Fragment Analyzer) before use in critical experiments, particularly for transfection or in vivo delivery applications.
Linear mRNA and circular RNA (circRNA) differ fundamentally in structure, stability, and translational mechanism. Linear mRNA carries a 5′ cap and poly(A) tail that enable efficient cap-dependent translation by the ribosome; it is ideal for studies requiring rapid, high-level transient protein expression, mRNA delivery research, and immunogen modeling. circRNA lacks free 5′ and 3′ ends, making it inherently resistant to exonucleolytic degradation, which confers substantially greater intracellular stability. Translation of circRNA occurs via internal ribosome entry sites (IRES) or other cap-independent mechanisms. circRNA is particularly valuable for miRNA sponge applications, sustained transgene expression platforms, and studies of RNA stability and function. Choose linear mRNA when fast, high-yield transient expression is needed; choose circRNA when extended intracellular stability, prolonged expression, or sponge-based loss-of-function studies are the priority.
Quality-controlled synthetic mRNA should be characterized by multiple orthogonal analytical methods. Standard assessments include: agarose gel electrophoresis or capillary electrophoresis (Bioanalyzer, Fragment Analyzer) to confirm full-length transcript integrity; HPLC to assess purity and residual double-stranded RNA (dsRNA) content; UV spectrophotometry for concentration and A260/A280 ratio; and optionally LC-MS for sequence and modification verification. A Certificate of Analysis (CoA) should accompany each lot, documenting yield, purity, integrity score, and endotoxin level (LAL assay) for products used in cell-based or animal studies. Functional activity is further confirmed by in vitro transfection followed by protein detection (e.g., flow cytometry, Western blot, or luminescence assay), confirming translational competence of the final product.
Synthetic mRNA can be delivered into cells and organisms through several established modalities. For in vitro applications, lipid-based transfection reagents (lipofection), electroporation, and polymer-based nanoparticles are the most common approaches. Lipid nanoparticles (LNPs) are the gold-standard delivery system and support high transfection efficiency both in vitro and in vivo. For in vivo studies, intramuscular, intravenous, intratumoral, or intraperitoneal administration may be used depending on the target tissue and research objective; the choice of delivery vehicle (LNP, polymeric carrier, or direct injection) should be matched to the organ of interest and application. Delivery efficiency is influenced by mRNA modifications, concentration, formulation composition, and cell type; optimization experiments are recommended for each new experimental system before scaling.
Can't find the mRNA or circRNA construct you need? We offer custom synthesis and add-on services to help you move your project forward — from sequence design and codon optimization to custom mRNA synthesis and circular RNA (circRNA) production for both in vitro and in vivo applications. Options may include chemically modified mRNA (e.g., N1-methylpseudouridine substitution, Cap1 capping strategy), circRNA synthesis via chemical ligation (short segments ≤100 nt) or IVT-based cyclization (longer constructs ≥200 nt), HPLC purification, and full QC documentation including gel or Bioanalyzer integrity analysis and a Certificate of Analysis. Additional options may include multiple synthesis scales from small research batches to larger quantities, miRNA sponge circRNA constructs, IRES element selection for cap-independent circRNA translation, labels and conjugation, and delivery formulation guidance. We can also assist with negative and scramble control formats and related RNA tools when a catalog product does not meet your specifications. Click Talk to a Scientist to submit a request, email us at support@biohippo.com, or explore our Research Services for additional support. Our team will be in contact with you shortly.
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