An optimized SpCas9 high-fidelity variant for direct protein delivery. Homo sapiens

The method of CRISPR-Cas delivery is crucial in determining editing efficacy and precision. Electroporation of the Cas9 ribonucleoprotein complex (RNP) offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity SpCas9 variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, here we developed a novel high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the new recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9) which is currently the only available high-fidelity Cas9 that can be used as RNP. The comparative analysis was extended to gene substitution experiments where the two high-fidelities were 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 throughout the tested genomic loci indicating different targeting capabilities between the two variants.The identification of a novel high-fidelity SpCas9 mutant, rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for highest precision and efficient applications.