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Cat. No.: eFrCas9-100 (for 100pmol)

Cat. No.: eFrCas9-500 (for 500pmol)

Cat. No.: eFrCas9-2500 (for 2500pmol)

Description

eFrCas9 is an engineered, enhanced version of FrCas9, developed through rational design based on cryo‑EM structural analysis of the FrCas9–sgRNA–DNA ternary complex combined with large‑scale mutational screening. By introducing targeted mutations into key residues within the phosphate lock loop and the PAM‑distal region, researchers significantly improved both the precision and efficiency of this Cas9 variant.

The phosphate lock loop is a critical structural element that regulates DNA binding and cleavage efficiency. Structural analysis revealed an electron‑density cavity at residue V1103. Substituting this site with lysine (V1103K) introduces a positively charged side chain that interacts more tightly with the DNA minor groove. This mutation increases editing efficiency by approximately 30% compared with wild‑type FrCas9, while reducing off‑target events by more than 30%.

Combining V1103K with an additional mutation in the PAM‑distal region (N732A) yields the eFrCas9 variant. In vitro cleavage assays show that eFrCas9 achieves 92.08% cleavage efficiency within just 0.25 minutes, while off‑target activity drops to 9.58%, demonstrating a substantial improvement in both speed and fidelity.

eFrCas9 retains the native FrCas9 PAM preference of 5'-NNTA-3', while the optimized phosphate lock loop enhances mismatch discrimination and the PAM‑distal modification improves overall DNA binding and cleavage kinetics. In a Duchenne muscular dystrophy (DMD) model, eFrCas9 successfully mediated precise deletions of 43.6–62.1 kb, achieving a maximum precise‑deletion efficiency of 17.25%, markedly outperforming SpCas9.