CRISPR-mediated directed codon re-write

A method for introducing a specific mutation at a specified position in a target gene of a cell of an organism, comprising:

• (a) introducing together, into a plurality of cells of the organism:
• an endonuclease protein;
• a first guide RNA that specifically recognizes a targeted position in the target gene;
• a second guide RNA that specifically recognizes a modified targeted position generated in step (i);
• and
• a third guide RNA that specifically recognizes a second modified targeted position generated in step (ii),
• (i) wherein the first guide RNA directs the nuclease to cleave the target gene at the targeted position to generate a first cleavage site, wherein a deletion of 2 or 3 nucleotides is introduced by repair at the first cleavage site to form a first modified targeted position;
• (ii) wherein the second guide RNA directs the nuclease to cleave the target gene at the first modified targeted position to generate a second cleavage site, wherein a first desired nucleotide being different from the original nucleotide is inserted by repair at this position, to form a second modified targeted position;
• (iii) wherein the third guide RNA directs the nuclease to cleave the target gene at the second modified targeted position to generate a third cleavage site, wherein a second desired nucleotide is inserted by repair at the third cleavage site to form a third modified targeted position, and wherein the third modified targeted position is further processed in step (iv) when the deletion in step (i) comprises 3 nucleotides;
• and
• (iv) when the deletion in step (i) comprises 3 nucleotides, introducing a fourth guide RNA that specifically recognizes the third modified targeted position, wherein the fourth guide RNA directs the nuclease to cleave the third modified targeted position to generate a fourth cleavage site, wherein a third desired nucleotide is inserted by repair at the fourth cleavage site to form a fourth modified targeted position;
• thereby resulting in a cell having a target gene in which two desired nucleotides replace two original nucleotides or three desired nucleotides replace three original nucleotides, without introducing a frameshift;
• and
• (b) optionally, screening the plurality of cells to isolate the cell in which the specific mutation has been introduced.