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 polynucleotide encoding a first selection marker operably linked to a first promoter and 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, and optionally wherein the first polynucleotide encoding the first selection marker is flanked by the first target site and the second target site; (b) introducing into the cell: (i) the first nuclease agent or a polynucleotide encoding 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 polynucleotide encoding a second selection marker operably linked to a second promoter and a second nuclease recognition site for a second nuclease agent, wherein the second nuclease recognition site is located within a coding region of the second selection marker or any non-protein-coding region of the second selection marker, wherein the first selection marker and the second selection marker are different, wherein the first nuclease agent is different from the second nuclease agent, 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, optionally wherein the identifying is carried out via a modification of allele assay. (a) providing the cell comprising the target locus, wherein the target locus comprises a first polynucleotide encoding a first selection marker operably linked to a first promoter and 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, and optionally wherein the first polynucleotide encoding the first selection marker is flanked by the first target site and the second target site; (b) introducing into the cell: (i) the first nuclease agent or a polynucleotide encoding 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 polynucleotide encoding a second selection marker operably linked to a second promoter and a second nuclease recognition site for a second nuclease agent, wherein the second nuclease recognition site is located within a coding region of the second selection marker or any non-protein-coding region of the second selection marker, wherein the first selection marker and the second selection marker are different, wherein the first nuclease agent is different from the second nuclease agent, and (i) the first nuclease agent or a polynucleotide encoding 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 polynucleotide encoding a second selection marker operably linked to a second promoter and a second nuclease recognition site for a second nuclease agent, wherein the second nuclease recognition site is located within a coding region of the second selection marker or any non-protein-coding region of the second selection marker, wherein the first selection marker and the second selection marker are different, wherein the first nuclease agent is different from the second nuclease agent, and wherein the first selection marker and the second selection marker are different, wherein the first nuclease agent is different from the second nuclease agent, 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, optionally wherein the identifying is carried out via a modification of allele assay.