Recombinogenic nucleic acid strands in situ

A method for modifying a target nucleic acid strand in vitro, comprising:

• (a) providing a nucleic acid vector comprising a first nucleic acid strand, wherein the first strand comprises:
• i. a first homology arm (HA1) that is homologous to a first nucleotide sequence (NS1) of a donor strand for homologous recombination between the first strand and the donor strand;
• ii. a second homology arm (HA2) that is homologous to a second nucleotide sequence (NS2) of the donor strand for homologous recombination between the first strand and the donor strand;
• and
• iii. an intervening nucleotide sequence joining the 3′ end of HA1 to the 5′ end of HA2;
• wherein HA1 is 5′ to HA2 in the first strand;
• and NS1 is 3′ to NS2 in the donor strand,
• wherein the donor strand comprises a retrieval sequence (RS) between NS1 and NS2;
• (b) combining the first strand with the donor strand;
• (c) performing Cas nuclease cutting of a first cut site (CS1) of the first strand at the 5′ end of HA1, or flanking 5′ of the 5′ end of HA1;
• and/or Cas nuclease cutting of a second cut site (CS2) of the first strand at the 3′ end of HA2, or flanking 3′ of the 3′ end of HA2 to provide a cut first strand;
• and
• wherein steps (b) and (c) are carried out in any order or simultaneously;
• (d) carrying out homologous recombination of the cut first strand with the donor strand, whereby gap repair of the cut first strand produces a product vector in which RS is retrieved between HA1 and HA2, and wherein the product of step (d) is a circular vector;
• and
• (e) inserting all or part of said RS of the product vector into the target nucleic acid strand;
• wherein a mutation of the target nucleic acid strand is removed by said insertion of RS into the target nucleic acid strand.