Population-Hastened Assembly Genetic Engineering

A directed nuclease for genome modification, comprising:

• a repeatable directed endonuclease, the repeatable directed endonuclease comprising:
• a nucleic acid recognition domain;
• a nucleic acid endonuclease domain;
• and
• a linker fusing or causing interaction between the nucleic acid recognition domain and the nucleic acid endonuclease domain, wherein the nucleic acid endonuclease creates a break in a target nucleic acid strand that is offset from the recognition sequence of the nucleic acid recognition domain.

A method for scalable multiplexed genome modification, the method comprising the steps of:

• providing a mixed population of viruses and cells, wherein at least some of the viruses lack a complete set of genes necessary for viral replication and instead encode for donor nucleotides, wherein at least some of the cells are transmitter cells that contain the genes needed for the viruses to replicate, and wherein at least some others of the cells are receiver cells containing the genome to be modified and any biochemical components necessary for modification of the genome by the donor nucleotides, including at least one engineered directed endonuclease, but do not contain the genes required for the viruses to replicate;
• causing at least some of the viruses to infect at least some of the transmitter cells and encode the donor nucleotides;
• modifying the genome in at least one receiver cell,
• wherein at least one engineered directed endonuclease in the receiver cell creates a break in a nucleic acid strand of the genome to be modified, wherein the engineered directed endonuclease comprises a nucleic acid recognition domain, a nucleic acid endonuclease domain, and a linker fusing or causing interaction between the nucleic acid recognition domain and the nucleic acid endonuclease domain, wherein the nucleic acid recognition domain of the engineered directed nuclease binds to a recognition sequence within the nucleic acid strand of the genome to be modified, the break in the nucleic acid strand of the genome to be modified being outside the recognition sequence of the nucleic acid strand;
• and wherein homologous recombination of the nucleic acid strand with a donor nucleotide received from a donor cell occurs to create a modified genome in the receiver cell;
• and
• replicating the modified genome in the receiver cell.