Methods and compositions for modifying a targeted locus

A method for serial modification of a target locus in a cell, comprising:

• (a) providing the cell comprising the target locus, wherein the target locus comprises a first selection cassette comprising:
• (1) a nucleic acid encoding a first selection marker;
• and (2) a first nuclease recognition site for a first nuclease agent, wherein the first nuclease recognition site is located in a coding region of the first selection marker or any non-protein-coding region of the first selection marker;
• (b) introducing into the cell:
• (i) the first nuclease agent, wherein the first nuclease agent induces a nick or a double-strand break at the first nuclease recognition site;
• and
• (ii) a first targeting vector comprising a first insert polynucleotide flanked by a first homology arm corresponding to a first target site located in the target locus and a second homology arm corresponding to a second target site located in the target locus, wherein the first insert polynucleotide comprises a second selection cassette comprising:
• (1) a nucleic acid encoding a second selection marker, wherein the first selection marker and the second selection marker are different;
• and (2) a second nuclease recognition site for a second nuclease agent, wherein the second nuclease recognition site is located in a coding region of the second selection marker or any non-protein-coding region of the second selection marker;
• and
• (c) identifying a modified cell comprising the first insert polynucleotide at the target locus, wherein the modified cell has the activity of the second selection marker but does not have the activity of the first selection marker;
• (d) introducing into the modified cell:
• (i) the second nuclease agent, wherein the second nuclease agent induces a nick or a double-strand break at the second nuclease recognition site;
• and
• (ii) a second targeting vector comprising a second insert polynucleotide flanked by a third homology arm corresponding to a third target site located in the target locus and a fourth homology arm corresponding to a fourth target site located in the target locus, wherein the second insert polynucleotide comprises a third selection cassette comprising:
• (1) a nucleic acid encoding a third selection marker, wherein the first selection marker and the third selection marker are identical;
• and (2) a third nuclease recognition site for a third nuclease agent, wherein the third nuclease recognition site is identical to the first nuclease recognition site and different from the second nuclease recognition site, and the first nuclease agent and the third nuclease agent are identical to one another and are different from the second nuclease agent;
• and
• (e) identifying at least one cell comprising the second insert polynucleotide integrated at the target locus, wherein the at least one cell has the activity of the third selection marker but does not have the activity of the second selection marker.