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

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.

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.

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.