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.
CRISPR-Cas9 has been demonstrated to be an effective gene editing tool used in therapeutics to save lives, but only a few Cas9 nucleases show exceptional activity from preclinical to clinical application. Introducing eSpOT-ON nuclease, 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.
| Engineered CRISPR Cas9 nuclease | Validated for cell and gene therapies or CRISPR-based therapies |
| High fidelity (high on-target; low off-target) | Easier DNA integration in the genome due to staggered cut editing |
| Simplified commercial sub-license available | Optimized chemically modified sgRNA for top performance |
What sets eSpOT-ON apart from other Cas9 protein is its exceptional combination of high on-target editing efficiency and reduced off-target activity. Through engineering, eSpOT-ON was designed to minimize unintended DNA cuts, significantly reducing off-target activity compared to standard or even other engineered high fidelity Cas9 protein variants. Additionally, eSpOT-ON has a reduced risk of chromosomal translocations, a critical safety concern in therapeutic gene editing. This makes eSpOT-ON the next-generation of CRISPR Cas9 protein optimized not only for accuracy but also for safety which is a key advantage for using in cell and gene therapies.
Unlike other Cas9 protein, eSpOT-ON is offered with a simplified sub-licensing model designed to reduce complexity and accelerate your path to market. With lower upfront costs and clearly defined milestones, you can focus more on advancing your cell and gene therapy and less on navigating licensing hurdles with our high fidelity Cas9 protein. This simplified license approach makes eSpOT-ON not only a scientifically advanced high fidelity Cas9 protein, but also a commercially accessible one.
Connect with our team to learn how eSpOT-ON can support your therapeutic pipeline with a licensing strategy that’s as efficient as eSpOT-ON itself empowering you to bring life-changing treatments to patients faster.
The engineered high fidelity Cas9 nuclease eSpOT-ON, delivered as high fidelity Cas9 protein, offers exceptional specificity with minimal off-target effects, as rigorously confirmed by CHANGE-seq analysis. Compared to standard Cas9 protein and HiFi Cas9 protein, our high fidelity Cas9 protein, eSpOT-ON, provides precise genome editing and dramatically reduces the risk of unintended DNA modifications, including a notably low rate of chromosomal translocations, which are critical safety concerns in therapeutic applications. These safety advantages position eSpOT-ON as a superior high fidelity Cas9 protein for clinical and translational research, ensuring safer and more reliable gene editing for cell and gene therapy development. When selecting a high fidelity Cas9 protein for therapeutic use, eSpOT-ON’s engineered high fidelity and safety profile make it the optimal choice for advancing your CRISPR-based therapies.
Compared to SpCas9, eSpOT-ON is unique in that it produces staggered DNA cuts, rather than blunt ends, during genome editing. This cutting pattern more effectively recruits the cell’s natural repair pathways, particularly those favoring precise integration of new genetic material. As a result, our high fidelity Cas9 protein enables more efficient and accurate DNA insertion, which is especially important for applications like targeted knock-ins or therapeutic gene correction. This engineered high fidelity Cas9 protein is optimized for precision, making it an ideal choice for a wide range of CRISPR-based gene editing applications, including cell and gene therapies.
To maximize the performance of eSpOT-ON, a next-generation engineered high fidelity Cas9 protein derived from PsCas9, the guide RNA (gRNA) was also carefully optimized through additional engineering. This involved removing unnecessary structural elements to enhance eSpOT-ON’s gRNA and target DNA binding affinity. Key chemical modifications, such as 2'-O-methyl analogs and 3' phosphorothioate linkages, were strategically incorporated to increase stability and reduce off-target effects. These refinements maximize the precision and efficiency of eSpOT-ON achieving top performance in gene editing, making it a powerful tool for advancing cell and gene therapy.
What are you waiting for? Switch to an engineered high fidelity Cas9 nuclease with higher fidelity today!
| 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% |
It is strongly encouraged that you work in an RNase-free and sterile environment when using eSpOT-ON nuclease and its sgRNA. Additionally, we strongly encourage you to use sterile filter pipette tips to decrease risk of introducing RNase or other contaminants into your samples. See our User Guide for our high fidelity Cas9 protein for more details on storage, handling, and user information.
eSpOT-ON nuclease |
SpCas9 nuclease |
||
|---|---|---|---|
| Size | 1409 amino acids | 1368 amino acids | |
| PAM Sequence (N = any nucleotide) |
5'-NGG-3' PAM is 3' of the target DNA sequence |
5'-NGG-3' PAM is 3' of the target DNA sequence |
|
| 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. |
Blunt end cut Cleavage 3 nt upstream of PAM sequence |
|
| Endonuclease Domains | HNH and RuvC | HNH and RuvC | |
| Full length gRNA | 109 nt | 97 - 103 nt | |
| Target Sequence | 22 nt | 20 nt | |
| gRNA Components | crRNA + tracrRNA as a single guide RNA (sgRNA) | crRNA + tracrRNA as a single guide RNA (sgRNA) | |
| Variants or Origin | Engineered from Parasutterella secunda Cas9 (PsCas9) | Streptococcus pyogenes Cas9 (SpCas9) | |
| CRISPR Enzyme Class | Type IIb CRISPR-Cas system of Parasutterella secunda Cas9 | Type IIa CRISPR-Cas system of Streptococcus pyogenes Cas9 |
Unsure which Cas nuclease is right for you? Explore our other cutting-edge nucleases to find the best fit for your CRISPR gene editing applications.
Part of eSpOT-ON nuclease engineering was optimizing its gRNA by adjusting its length and incorporating chemical modifications at strategic locations to enhance binding affinity and peformance. Through the gRNA chemical modification optimization for our engineered high fidelity Cas9 protein, eSpOT-ON achieved the best editing efficiency and minimized off-targets using the following chemical modifications on its full-length sgRNA sequence:
2'-O-Methyl analog at the first 3 and last 3 bases and 3' phosphorothioate bonds between 3 first and last 4 bases
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 tools such as CHOPCHOP, Benchling, CRISPOR, or CRISPR RGEN Cas-Designer Tool. 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.
Once you’ve received your eSpOT-ON protein and gRNA, we highly recommend using our step-by-step eSpOT-ON Protein and gRNA User Guide to ensure a smooth and successful start to your CRISPR experiments. We also adapted a variety of protocols designed specifically for our nucleases and gRNA, including nucleofection, electroporation, and lipofection. After transfection, you can follow our genotyping protocol and use platforms like Inference of CRISPR Edits, ICE for short, to help you analyze your success.
Challenge: Low-performing Cas9 nucleases often result in minimal or no editing across many gene targets, making it difficult for scientists to generate reproducible results. In critical applications like CRISPR-based cell and gene therapies, this lack of editing efficiency can jeopardize therapeutic success where precision and performance are essential.
Solution: The engineered high fidelity Cas9 nuclease, eSpOT-ON, consistently delivers robust editing across a wide range of gene targets. In many cases, it matches or exceeds the performance of other Cas9 variants, enabling scientists to achieve reliable and efficient editing where it matters most.
Challenge: Some engineered HiFi / high fidelity Cas9 protein lack precision resulting in the binding and cutting of unintended genomic sites, leading to off target edits. These unwanted edits may trigger toxic effects, raise serious safety concerns, and slow down key milestones in therapeutic development.
Solution: Engineered for precision, eSpOT-ON outperforms other high fidelity Cas9 protein (HiFi Cas9 protein), delivering better safety and reliability for gene editing in therapeutic applications.
eSpOT-ON is an engineered high fidelity Cas9 nuclease (ePsCas9) designed for high-efficiency, In vivo genome editing. Developed through structural analysis and rational protein engineering, eSpOT-ON nuclease demonstrates significantly enhanced editing activity across multiple gene targets while maintaining strong safety characteristics, including minimal off-target effects and a low rate of chromosomal translocations compared to other high fidelity Cas9 protein on the market. When delivered using lipid nanoparticles, eSpOT-ON nuclease successfully edited the Pcsk9 gene in mouse liver, showcasing its potential for non-viral therapeutic applications such as treating genetic diseases like hypercholesterolemia. This Nature Communications publication walks you through the engineering and data, showcasing why eSpOT-ON, our high fidelity Cas9 nuclease, was designed for therapeutic applications.
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.
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