CAS9MODIFICADA MOLECULE ALONE， CAS9MODIFICADA， METHOD FOR PRODUCING A PROTEIN OR RIBONUCLEOPROTE??NA COMPLEXES OR COMPLEXES OF PROTEIN and RIBONUCLEOPROTE??NA MIXED OR LIP??DEO， FUSION PROTEIN， NUCLEOTIDE SEQUENCE， NUCLEIC ACID， VECTOR， CELL， ANIMAL OR PLANT， PHARMACEUTICAL COMPOSITION， IN VITRO USE OF A RECOMBINANT CAS9MODIFICADA， KIT OF PARTS， and IN VITRO METHOD FOR ALTERING THE GENOME OF A CELL

To address the limitations deriving from the unspecific genomic cleavages of the Streptococcus pyogenes Cas9 (SpCas9) and to identify variants with higher cleavage fidelity， the present invention describes a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets. The screening of SpCas9 variants obtained by random mutagenesis of the Red -II domain allowed the identification of hits with increased on/off ratios. The best performing nuclease， evoCas9， was isolated through the combination of the identified mutations within a single variant. Side by side analyses with previously reported rationally designed variants demonstrated a significant improvement in fidelity of evoCas9 of the present invention. To address the limitations deriving from the unspecific genomic cleavages of the Streptococcus pyogenes Cas9 (SpCas9) and to identify variants with higher cleavage fidelity， the present invention describes a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets. The screening of SpCas9 variants obtained by random mutagenesis of the Red -II domain allowed the identification of hits with increased on/off ratios. The best performing nuclease， evoCas9， was isolated through the combination of the identified mutations within a single variant. Side by side analyses with previously reported rationally designed variants demonstrated a significant improvement in fidelity of evoCas9 of the present invention.