Methods for genomic modification
AssigneeAmyris, Inc.
InventorsZach Serber, Andrew Horwitz
Life of this patent
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Abstract
Provided herein are methods of integrating one or more exogenous nucleic acids into one or more selected target sites of a host cell genome. In certain embodiments, the methods comprise contacting the host cell genome with one or more integration polynucleotides comprising an exogenous nucleic acid to be integrated into a genomic target site, and a nuclease capable of causing a double-strand break near or within the genomic target site.
Claims78
A method for simultaneously integrating a plurality of (n) exogenous nucleic acids into a plurality of (n) target sites of a host cell genome, wherein n is at least two, the method comprising:
- (a) simultaneously contacting a host cell with:
- (i) said plurality of exogenous nucleic acids, wherein:
- x is an integer that varies from 1 to n, and for each integer x, each exogenous nucleic acid (ES)x comprises a first homology region (HR1)x and a second homology region (HR2)x, wherein (HR1)x and (HR2)x are capable of initiating host cell mediated homologous recombination of (ES)x at a target site (TS)x selected from said plurality of (n) target sites of said host cell genome;
- and
- (ii) for each said target site (TS)x, a nuclease (N)x capable of cleaving at (TS)x whereupon said cleaving results in homologous recombination of (ES)x at (TS)x;
- and
- (b) recovering a host cell wherein each exogenous nucleic acid (ES)x has integrated at its selected target site (TS)x,
- wherein said recovering occurs at a frequency of about one every 10, 9, 8, 7, 6, 5, 4, 3, or 2 contacted host cells, or clonal populations thereof, screened.
A method for integrating a plurality of exogenous nucleic acids into a host cell genome, the method comprising:
- (a) contacting a host cell with:
- (i) a plurality of exogenous nucleic acids, wherein:
- for each integer x, each exogenous nucleic acid (ES)x comprises a first homology region (HR1)x and a second homology region (HR2)x, wherein (HR1)x and (HR2)x are capable of initiating host cell mediated homologous recombination of (ES)x at a target site (TS)x selected from a plurality of target sites of said host cell genome;
- and
- (ii) for each said target site (TS)x, a nuclease (N)x capable of cleaving at (TS)x, whereupon said cleaving results in homologous recombination of (ES)x at (TS)x;
- and
- (b) recovering a host cell wherein each exogenous nucleic acid (ES)x has integrated at its selected target site (TS)x,
- wherein said recovering occurs at a frequency of about one every 10, 9, 8, 7, 6, 5, 4, 3, or 2 contacted host cells, or clonal populations thereof, screened.
Dependencies are inferred from the claim text.