METHODS FOR CLONING AND MANIPULATING GENOMES

摘要

Compositions and methods are disclosed herein for cloning a synthetic or a semi-synthetic donor genome in a heterologous host cell. In one embodiment, the donor genome can be further modified within a host cell. Modified or unmodified genomes can be further isolated from the host cell and transferred to a recipient cell. Methods disclosed herein can be used to alter donor genomes from intractable donor cells in more tractable host cells.

主权项

1. A method for seamlessly introducing a modification in a target nucleic acid molecule present in a host cell, comprising:
a. introducing a mutagenesis construct and a host vector into the host cell whereby the host vector recombines with the mutagenesis construct in the host cell, wherein the mutagenesis construct contains
a first portion of homology to a 5′ portion of the target nucleic acid molecule upstream of the modification;an endonuclease recognition site;a promoter;a gene encoding the endonuclease; anda selectable marker;a second repeat portion of homology that is homologous to a sequence of the target nucleic acid molecule upstream of a target region on the target nucleic acid molecule;a third portion of homology that is homologous to a 3′ portion of the target region on the target nucleic acid molecule downstream of the modification; and b. incubating the cells under conditions
i. whereby recombination occurs between the first portion of homology and the upstream or downstream portion, thereby seamlessly removing a portion of the construct,ii. that promote one or more double-strand break cleavages in the target nucleic acid molecule near the target region containing the construct, whereby a modification is seamlessly introduced into the target nucleic acid molecule.