An optimized SpCas9 high-fidelity variant for direct protein delivery

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and po-tential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fi-delity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible withRNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNPdelivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D sub-stitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 thatcan be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelitieswere used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ)versus homology-directed repair (HDR) for precise editing.The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two var-iants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currentlyused HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.