eFrCas9 Enhanced Nuclease

Product Introduction

eFrCas9 is an engineered enhanced version of FrCas9, a high-precision, high-efficiency Cas9 variant developed through rational design based on cryo-EM structural analysis of the FrCas9-sgRNA-DNA ternary complex and large-scale mutagenesis screening. The research team performed targeted mutagenesis on key residues in the phosphate lock loop and PAM-distal region of FrCas9, synergistically improving its editing precision and efficiency.

The phosphate lock loop is a key element regulating DNA binding and cleavage efficiency. Structural analysis revealed an electron density cavity at the V1103 site within this region. Substituting it with lysine (V1103K) allowed its positively charged side chain to bind more tightly to the DNA minor groove, increasing editing efficiency by approximately 30% compared to wild-type FrCas9 while reducing off-target events by over 30%. By further combining V1103K with the N732A mutation in the PAM-distal region, the resulting eFrCas9 variant achieved 92.08% cleavage efficiency within 0.25 minutes in in vitro cleavage assays, with off-target activity reduced to 9.58%.

eFrCas9 retains the wild-type FrCas9 5'-NNTA-3' PAM recognition characteristic, while the optimization of the phosphate lock loop enhances its ability to discriminate against mismatches. The modification of the PAM-distal region improves the overall binding and cleavage kinetics of the target DNA. In a Duchenne muscular dystrophy (DMD) model, eFrCas9 successfully achieved precise deletion of large fragments ranging from 43.6 to 62.1 kb, with precise deletion efficiencies reaching up to 17.25%, significantly outperforming SpCas9.