Methods for scarless introduction of targeted modifications into targeting vectors

A method for introducing a scarless targeted genetic modification in a preexisting targeting vector, comprising:

• (a) performing bacterial homologous recombination between the preexisting targeting vector and a modification cassette in a population of bacterial cells,
• wherein the modification cassette comprises the targeted genetic modification and comprises an insert nucleic acid flanked by a 5′ homology arm corresponding to a 5′ target sequence in the preexisting targeting vector and a 3′ homology arm corresponding to a 3′ target sequence in the preexisting targeting vector, wherein the insert nucleic acid comprises from 5′ to 3′:
• (i) a first repeat sequence;
• (ii) a first target site for a first nuclease agent;
• (iii) a selection cassette;
• (iv) a second target site for a second nuclease agent;
• and
• (v) a second repeat sequence identical to the first repeat sequence,
• wherein the repeat sequence is at least about 20 nucleotides in length;
• (b) selecting bacterial cells comprising a modified targeting vector comprising the selection cassette;
• (c) cleaving the first target site in the modified targeting vector with the first nuclease agent and cleaving the second target site in the modified targeting vector with the second nuclease agent to remove the selection cassette and expose the first repeat sequence and the second repeat sequence in the modified targeting vector,
• wherein step (c) occurs in vitro;
• and
• (d) assembling the exposed first repeat sequence with the exposed second repeat sequence in an intramolecular in vitro assembly reaction to generate the targeting vector comprising the scarless targeted genetic modification,
• wherein neither the first target site for the first nuclease agent nor the second target site for the second nuclease agent are present and only a single copy of the repeat sequence is present in the targeting vector comprising the scarless targeted genetic modification.