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ISOLATED MODIFIED CAS9 MOLECULE, METHOD TO PRODUCE A MODIFIED CAS9, PROTEIN OR RIBONUCLEOPROTEIN COMPLEXES OR COMPLEXES OF MIXED OR LIPID, COMPOSITE, LIPID, FUSION PROTEIN, NUTRITION, NUTRITION, NUTRITION IN VITRO USE OF A MODIFIED RECOMBINANT CAS9, PARTS KIT, AND, IN VITRO METHOD TO CHANGE A CELL GENOME
ISOLATED MODIFIED CAS9 MOLECULE, METHOD TO PRODUCE A MODIFIED CAS9, PROTEIN OR RIBONUCLEOPROTEIN COMPLEXES OR COMPLEXES OF MIXED OR LIPID, COMPOSITE, LIPID, FUSION PROTEIN, NUTRITION, NUTRITION, NUTRITION IN VITRO USE OF A MODIFIED RECOMBINANT CAS9, PARTS KIT, AND, IN VITRO METHOD TO CHANGE A CELL GENOME
to address the limitations arising from nonspecific genomic cleavages of the cas9 of streptococcus pyogenes (spcas9) and to identify variants with greater cleavage fidelity, the present invention describes a yeast-based assay that allows the simultaneous assessment of targeted and off-target activity to two engineered genomic targets. the selection of spcas9 variants obtained by random mutagenesis of the red-ii domain allowed the identification of hits with greater reasons on the target / off target. the best performing nuclease, evocas9, was isolated by combining the mutations identified in a single variant. side-by-side analyzes with rationally designed variants previously reported demonstrated a significant improvement in the fidelity of evocas9 of the present invention.
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