PRODUCT

eSpOT-ON mRNA

High-fidelity Cas9 mRNA for therapeutic gene editing.

eSpOT-ON is an engineered high-fidelity Cas9 variant, designed for therapeutic gene editing. Delivered in mRNA format, eSpOt-ON provides superior on-target activity with low off-target effects. This nuclease has been validated in preclinical studies and is ready to support your next breakthrough in cell and gene therapy development.

Engineered for precise and safe genome editing.
Significantly reduces off-target editing.
Lowers the risk of chromosomal translocations.
Validated in a wide range of human cell lines, including iPSCs.

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eSpOT-ON mRNA (100 µg)

#M20ESPOTON-Sm

Overview Specifications Data Resources

Overview

Engineered high fidelity Cas9 mRNA for cell and gene therapies

CRISPR-Cas9 has become a cornerstone of gene editing in therapeutic development, yet only a few nucleases deliver the fidelity and efficiency needed to succeed from early discovery to clinical applications. eSpOT-ON nuclease is a high fidelity Cas9 engineered from Parasutterella secunda Cas9 (PsCas9), also known as ePsCas9. As part of the Type IIb CRISPR-Cas system, eSpOT-ON sets a new standard in genome editing with unmatched specificity and robust activity. Our high fidelity Cas9 mRNA, eSpOT-ON is ready to be integrated in your mRNA based therapeutic pipeline to save patient lives.

Benefits of using eSpOT-ON nuclease in your therapeutic pipeline

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

Engineered high fidelity Cas9 mRNA for precise and safe genome editing

We also offer eSpOT-ON is an engineered high fidelity Cas9 mRNA designed to deliver superior on-target editing efficiency while drastically minimizing off-target effects. Unlike conventional Cas9 nucleases, our high fidelity Cas9 nuclease has been optimized through precise engineering to enhance genome editing specificity. The result is eSpOT-ON, a high fidelity Cas9 mRNA that significantly reduces unintended DNA cleavage, making it ideal for applications where accuracy is paramount, especially in therapeutic gene editing. With safety being top of mind, this high fidelity Cas9 mRNA also lowers the risk of chromosomal translocations, addressing key safety concerns in cell and gene therapy other Cas9 mRNA might impose. With its balance of accuracy and precision, eSpOT-ON sets a new benchmark among engineered HiFi Cas9 mRNA nucleases used for clinical applications.

Licensing for engineered high fidelity Cas9 mRNA

eSpOT-ON is more than just a high-performance engineered high fidelity Cas9 mRNA. It’s also available through a simplified commercial sub-license that eliminates many of the common barriers to CRISPR-based therapeutic development. Unlike traditional licensing frameworks associated with standard or HiFi Cas9 nucleases, our model reduces upfront costs, defines clear development milestones, and helps you move faster toward clinical and commercial success. This straightforward path to success makes eSpOT-ON not only a cutting-edge high fidelity Cas9 mRNA tool for precise genome editing, but also a commercially viable solution for scientists advancing cell and gene therapies.

Connect with our team to learn how licensing eSpOT-ON, our engineered high fidelity Cas9 mRNA, can accelerate your cell and gene therapy development. With a streamlined path to commercialization, eSpOT-ON empowers you to advance cutting-edge therapies with both scientific precision and commercial efficiency.

Clinically validated high fidelity Cas9 mRNA for therapeutic genome editing

Extensive validation of eSpOT-ON delivered as high fidelity Cas9 mRNA demonstrates consistent, robust genome editing across a wide range of human cell lines, including HEK293T, HeLa, Huh7, DLD-1, and induced pluripotent stem cells (iPSCs). This engineered high fidelity Cas9 mRNA also shows precise on-target activity at clinically relevant genes such as PCSK9, TRAC, and EMX1, critical for therapeutic gene editing applications. Combined with its strong compatibility with lipid nanoparticle (LNP) delivery systems, eSpOT-ON represents a powerful and reliable high fidelity Cas9 mRNA nuclease for advancing CRISPR-based cell and gene therapies.

Enhanced genome editing with engineered high fidelity Cas9 mRNA producing staggered DNA cuts

Unlike conventional Cas9 nucleases that create blunt DNA breaks, eSpOT-ON produces staggered DNA cuts, which significantly enhance the cell’s natural DNA repair mechanisms. This results in improved accuracy and efficiency of DNA integration, especially important when using our high fidelity Cas9 mRNA for targeted gene knock-ins or therapeutic gene correction. By leveraging our high fidelity Cas9 mRNA, eSpOT-ON, scientists can achieve highly specific editing with minimal off-target effects. eSpOT-ON’s unique design makes it the ideal Cas9 mRNA choice for advanced CRISPR applications in cell and gene therapy.

Chemically modified sgRNA optimized for maximum performance with engineered high fidelity Cas9 mRNA, eSpOT-ON

To unlock the full potential of eSpOT-ON, our engineered high fidelity Cas9 mRNA, its single guide RNA (sgRNA) has been precisely optimized for superior gene editing efficiency. By removing unnecessary structural components and incorporating advanced chemical modifications like 2'-O-methyl analogs and 3' phosphorothioate bonds at key positions, this chemically modified sgRNA enhances the stability and specificity of the eSpOT-ON. These enhancements drive the exceptional precision and efficiency of our high fidelity Cas9, eSpOT-ON, in a variety of applications including cutting-edge cell and gene therapies.

Don’t wait! Upgrade to an engineered high fidelity Cas9 mRNA nuclease today and experience unmatched precision for your therapeutic pipeline.

eSpOT-ON Origin	Engineered from Parasutterella secunda (PsCas9)
Molecular weight	165 ng/pmol
Concentration	1 µg/ul
eSpOT-ON mRNA Amount Offering	100 µg 500 µg 1 mg 5 mg
eSpOT-ON mRNA Fill Volume	100 µL for 100 µg 500 µL for 500 µg 1000 µL for 1 mg 5000 µL for 5 mg
eSpOT-ON mRNA Storage Buffer	10 mM Tris-HCl (pH 7.5), 1 mM EDTA, RNase-free
eSpOT-ON mRNA Storage Conditions	Recommended storage at -80±10°C
eSpOT-ON mRNA Shipping conditions	Shipped frozen on dry ice
eSpOT-ON mRNA Stability	At storage temperature at -80±10°C, the shelf life is indicated by the expiration date printed on the vial label.

Recommendations for handling eSpOT-ON nuclease mRNA

It is strongly encouraged that you work in an RNase-free and sterile environment when using eSpOT-ON nuclease mRNA and its gRNA. Additionally, we strongly encourage you to use sterile filter pipette tips to decrease risk of introducing RNase or other contaminants into your samples. Read more about how to use our high fidelity Cas9 mRNA in its User Guide. It dives into storage recommendations, handling suggestions, and more for eSpOT-ON nuclease mRNA.

What's the difference between eSpOT-ON and SpCas9 nuclease?

RNP complex schematics of eSpOT-ON (left) and SpCas9 (right). Depicted in the schematic are genomic DNA (light blue), PAM sequence (pink), and predicted cut site locations on genomic DNA (gray scissors). The guide RNA (gRNA) is composed of the target sequence (green) and scaffold (dark blue).

	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

Not sure which nuclease suits your needs? Discover our full nuclease portfolio of cutting-edge nucleases to find the ideal match for your CRISPR gene editing therapeutic pipeline.

Data

Designing optimized gRNA for eSpOT-ON nuclease

How eSpOT-ON gRNA were engineered

As part of the development of eSpOT-ON nuclease, the guide RNA (gRNA) was carefully engineered for optimal performance. This included fine-tuning gRNA length and introducing strategic chemical modifications to enhance binding affinity to the target, boosting eSpOT-ON's editing efficiency, and reduce off-target effects. Through systematic optimization of these chemical modifications on the gRNA, our engineered high fidelity Cas9 mRNA, eSpOT-ON, delivers exceptional on-target activity. The optimized full-length gRNA includes the following modifications:

2'-O-Methyl analog at the first 3 and last 3 bases and 3' phosphorothioate bonds between 3 first and last 4 bases

Tips for designing gRNA for your eSpOT-ON nuclease

Designed for use with eSpOT-ON mRNA, the guide RNA full-length sequence is a 109 nucleotide long that includes a 22 nucleotide target sequence. When eSpOT-ON is complexed with its gRNA, it creates precise staggered DNA cuts cleaving the target strand 3 nucleotides upstream of the PAM and the non-target strand 6–7 nucleotides upstream. To design guide RNAs compatible with eSpOT-ON mRNA, you can use tools such as CHOPCHOP, Benchling, CRISPOR, or CRISPR RGEN Cas-Designer Tool. If eSpOT-ON isn’t listed as a default option, simply input the 5’-NGG-3’ PAM and its corresponding staggered cut profile to design your eSpOT-ON gRNA. Once you’ve selected your target sequence, paste it into our ordering platform. Our team will incorporate the correct eSpOT-ON scaffold (constant region) during synthesis to deliver a complete, ready-to-use gRNA.

To support the seamless integration of our high fidelity Cas9 mRNA into your CRISPR workflows, we offer the eSpOT-ON Controls Kit, a convenient way to evaluate performance before moving to your specific targets. The kit includes two validated human positive control sgRNAs and one non-targeting control, providing a reliable benchmark for testing the efficiency and specificity of this high-fidelity engineered Cas9.

Delivering eSpOT-ON mRNA and gRNA, analyzing edits

After receiving your eSpOT-ON mRNA and gRNA, we recommend starting with our detailed eSpOT-ON mRNA and gRNA User Guide to ensure a smooth and successful start to your CRISPR experiments. We've also developed a variety of optimized protocols explicitly designed for our nucleases and guide RNAs, including protocols for nucleofection, electroporation, and lipofection. Following transfection, you can assess CRISPR editing results using our genotyping protocols and tools like ICE (Inference of CRISPR Edits) to evaluate your results with confidence.

Engineered high fidelity Cas9 mRNA designed for therapeutic applications

Challenge: Using low fidelity Cas9 mRNA in cell and gene therapies leads to reduced editing efficiency, increased off-target activity, and diminished therapeutic effectiveness. Even more concerning, these nucleases heighten safety risks, hindering the successful progression of therapies through clinical trials.

Solution: In therapeutic applications, eSpOT-ON mRNA, our engineered high fidelity Cas9 mRNA, delivers precise gene editing with consistent, desired outcomes all while maintaining a high safety profile - what you look for in a nuclease to reach clinical trials.

Proven in vivo success with high fidelity Cas9 mRNA in therapeutic applications

Schematic of a *in vivo* preclinical study to evaluate the editing capacity of SpCas9 and eSpOT-ON in mice delivered as Cas9 mRNA.

*In vivo* genome editing activity of SpCas9 and eSpOT-ON delivered as Cas9 mRNA in preclinical studies.

Plasma Pcsk9 levels in mouse plasma post genome editing with SpCas9 and eSpOT-ON. Both nucleases were delivered in the form of Cas9 mRNA during *In vivo* preclinical studies.

A) Mouse body weight measurements during *In vivo* preclinical studies with SpCas9 and eSpOT-ON, delivered as Cas9 mRNA. B) Activity of alanine transaminase (ALT) and aspartate aminotransferase (AST) in liver 7 days after LNP dosing demonstrating the safety of our engineered high fidelity Cas9 mRNA nuclease, eSpOT-ON.

Reliable Cas9 mRNA editing performance: eSpOT-ON excels in multiple cell types

Gene editing activity of eSpOT-ON mRNA and SpCas9 mRNA evaluated at 5 target sites across the genome in HEK293T, HeLa, Huh7, DLD-1 and iPSC cells mediated via simultaneous delivery and its respective gRNA. Data are shown as mean ± SD for n=3 biological repeats. Our high fidelity Cas9 mRNA demonstrates robust gene editing across a variety of cell lines and targets.

RESOURCES

If you like eSpOT-ON nuclease, explore our other resources:

eSpOT-ON nuclease protein and gRNA User Guide.

CRISPR Clinical Trials to Follow.

Synthetic sgRNAs Enable Researchers to Study Viral Infection in Resting Human CD4+ T Cells.

Explore our protocols

Do you want to see more data about eSpOT-ON nuclease and its performance as a high fidelity Cas9 mRNA in therapeutic applications?

eSpOT-ON, an engineered high fidelity Cas9 nuclease, aka ePsCas9, was optimized for precise and efficient genome editing for therapeutic applications. Engineered through rational nuclease design and guided by structural insights, as a high fidelity Cas9 nuclease, eSpOT-ON delivers robust editing performance across diverse gene targets while upholding a strong safety profile marked by low off-target activity and a reduced incidence of chromosomal translocations when compared to other high fidelity Cas9 nucleases. In preclinical studies using lipid nanoparticle delivery, eSpOT-ON mRNA enabled effective editing of the Pcsk9 gene in the liver of mice, highlighting its promise for non-viral gene editing strategies in therapeutic areas such as inherited metabolic disorders. The foundational research published in Nature Communications details the engineering process and in vivo results that position eSpOT-ON as a leading candidate for therapeutic genome editing.

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.