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The following modifications are included on our eSpOT-ON modified gRNA:
2'-O-Methyl analog at the first 3 and last 3 bases and 3' phosphorothioate bonds between 3 first and last 4 bases
High-fidelity Cas9 protein for precise gene editing.
eSpOT-ON nuclease is an engineered high-fidelity Cas9 protein designed for therapeutic applications. It offers high on-target editing efficiency while minimizing off-target activity, making it a safer choice for precise cell and gene therapy development. This Cas9 protein is optimized for both accuracy and safety, providing a reliable solution for your gene editing needs.
eSpOT-ON is an engineered high-fidelity Cas9 nuclease derived from Parasutterella secunda Cas9 (PsCas9) and also known as engineered PsCas9 (ePsCas9). This engineered high fidelity Cas9 nuclease belongs to the Type IIb CRISPR-Cas system and provides the specificity and activity needed to drive your therapeutic success.
It delivers precise, high on-target activity with minimal off-target effects, generates staggered DNA cuts that support efficient DNA integration, and is validated across multiple human cell types. With strong compatibility for LNP delivery and access through a simplified commercial sublicense, eSpOT-ON provides a clinically aligned Cas9 protein solution for advancing cell and gene therapies.
High-Fidelity Editing:
Maximizes on-target activity while minimizing off-target modifications.
Staggered DNA Breaks:
Supports efficient and precise DNA integration for knock-ins or gene corrections.
Validated Across Cell Types:
Demonstrated performance in HEK293T, HeLa, Huh7, DLD-1, and iPSCs.
Optimized sgRNA Compatibility:
Chemically modified sgRNA enhances stability and genome targeting.
Simplified Licensing:
Streamlined commercial sublicense accelerates therapeutic development.
| Product Number | R20ESPOTON |
| Concentration | 10 mg/ml |
| Delivery Format | 1.5 ml plastic tube 5 x 1.5ml plastic tubes |
| Intended Use | This product is intended for research use only |
| Shipping | Cold Pack |
| Buffer Composition | 20 mM HEPES, 300 mM NaCl, 50% Glycerol, 1 mM DTT, 1 mM EDTA, pH 7.5 |
| Source | E. coli |
| Purity | ≥ 95.0% |
| Specification | eSpOT-ON |
|---|---|
| Size | 1409 amino acids |
| PAM Sequence (N = any nucleotide) | 5'-NGG-3’ |
| DNA Cleavage | Staggered-cut. Cleavage on the target strand occurs 3 nt upstream of the PAM, while the non-target strand is cut 6-7 nt upstream of the PAM. |
| Endonuclease Domains | HNH and RuvC |
| gRNA Length | 109nt |
| Target Sequence Length | 22 nt |
| Enzyme Class | Type IIb CRISPR-Cas system of Parasutterella secunda Cas9 |
With a 109 nucleotides (nt) guide sequence and 22nt target sequence, eSpOT-ON performs staggered DNA cuts, cleaving the target strand 3nt upstream of the PAM and the non-target strand 6-7nt upstream.
To design eSpOT-ON guide RNAs, you can use one of these guide design platforms. If the design tool doesn’t include eSpOT-ON by default, you can manually input the 5’-NGG-3’ PAM along with its staggered cut pattern to generate your eSpOT-ON guide RNAs targeting your region of interest. After selecting your eSpOT-ON gRNA target sequence, simply copy and paste it into our order platform and our team will take care of adding the eSpOT-ON scaffold (constant region) during synthesis.
To help you seamlessly integrate this high fidelity engineered Cas9 protein into your CRISPR workflows, we also offer the eSpOT-ON Controls Kit, Human. This eSpOT-ON Controls Kit includes two validated human positive control gRNAs and one non-targeting gRNA control, giving you a reliable and efficient way to test our high fidelity Cas9 protein in your experiments.
The following modifications are included on our eSpOT-ON modified gRNA:
2'-O-Methyl analog at the first 3 and last 3 bases and 3' phosphorothioate bonds between 3 first and last 4 bases
Low-performing Cas9 nucleases can limit editing efficiency across gene targets, making reproducibility difficult—especially in applications where precision is critical. eSpOT-ON is an engineered high-fidelity Cas9 protein designed to deliver consistent, efficient on-target editing across a broad range of targets. In many use cases, eSpOT-ON matches or exceeds the performance of other Cas9 variants, supporting dependable editing outcomes for demanding research and therapeutic workflows.
Some high-fidelity Cas9 proteins still exhibit reduced target specificity, leading to unintended binding and cleavage at off-target genomic sites. These off-target edits can introduce toxic effects, compromise cell viability, and create safety concerns that delay therapeutic development.
eSpOT-ON is an engineered high-fidelity Cas9 protein designed to improve target specificity and minimize off-target activity. Compared to other HiFi Cas9 proteins, eSpOT-ON delivers more controlled and predictable editing, supporting safer genome editing workflows for cell and gene therapy applications.
If you have additional questions please connect with a member of our team.
Because it produces staggered DNA cuts rather than blunt ends, eSpOT‑ON can also improve the efficiency and precision of DNA integration, which is beneficial for knock‑in, gene correction, or other insertion‑based edits.
eSpOT-ON mRNA delivers the genetic blueprint for the nuclease, which is translated by the cell after delivery. This format is well-suited for in vivo delivery approaches.
Both formats provide high-fidelity Cas9 editing; the optimal choice depends on your delivery method, desired editing kinetics, and experimental or therapeutic development needs.
Legal Terms: eSpOT-ON Nuclease purchased from Synthego is only intended and licensed to be used solely with eSpOT-ON guide RNAs purchased from Synthego, and eSpOT-ON guide RNAs purchased from Synthego are only intended and licensed to be used solely with eSpOT-ON Nuclease purchased from Synthego. For more details, visit our patent page.