Back to results
US 2017/0283779 A1Published application
Bacteriophage engineering methods
AssigneeThe Charles Stark Draper Laboratory, Inc.
InventorsJASON HOLDER, Connor McBrine, Sarah Gruszka, Miles Rogers, Nicole Billings
Life of this patent
Estimating the term…
Abstract
The present disclosure provides methods and kits for generating recombinant bacteriophage genomes.
Claims42
A method for generating a recombinant bacteriophage genome comprising:
- (a) contacting a non-recombinant bacteriophage genome with a sgRNA-CRISPR enzyme conjugate in vitro under conditions where
- the sgRNA-CRISPR enzyme conjugate cleaves a protospacer sequence within the non-recombinant bacteriophage genome to produce a cleaved non-recombinant bacteriophage genome;
- and
- (b) recombining in vitro the cleaved non-recombinant bacteriophage genome with a heterologous nucleic acid in the presence of a recombination system under conditions to produce a recombinant bacteriophage genome.
1 more element
A method for generating a recombinant bacteriophage genome comprising:
- (a) contacting a non-recombinant bacteriophage genome comprising a single first recognition site with a first restriction enzyme in vitro under conditions where the first restriction enzyme cleaves the first recognition site to produce a cleaved non-recombinant bacteriophage genome;
- and
- (b) recombining in vitro the cleaved non-recombinant bacteriophage genome with a heterologous nucleic acid in the presence of a recombination system under conditions to produce a recombinant bacteriophage genome.
A method for generating a recombinant bacteriophage genome comprising:
- (a) contacting a non-recombinant bacteriophage genome with
- (i) a sgRNA-CRISPR enzyme conjugate in vitro under conditions where the sgRNA-CRISPR enzyme conjugate cleaves a protospacer sequence within the non-recombinant bacteriophage genome to produce a cleaved non-recombinant bacteriophage genome;
- or
- (ii) a restriction enzyme in vitro under conditions where the restriction enzyme cleaves a unique recognition site within the non-recombinant bacteriophage genome to produce a cleaved non-recombinant bacteriophage genome;
- (b) transforming the cleaved non-recombinant bacteriophage genome into a bacterial host cell, wherein the bacterial host cell comprises a vector that expresses a heterologous nucleic acid;
- and
- (c) recombining in vivo the cleaved non-recombinant bacteriophage genome with the heterologous nucleic acid in the presence of a non-endogenous recombination system under conditions to produce a recombinant bacteriophage genome.
4 more elements
A method for generating a recombinant bacteriophage genome comprising:
- (a) transforming an intact non-recombinant bacteriophage genome into a bacterial host cell, wherein the bacterial host cell comprises a vector that expresses a heterologous nucleic acid;
- and
- (b) recombining in vivo the intact non-recombinant bacteriophage genome with the heterologous nucleic acid in the presence of a non-endogenous recombination system under conditions to produce a recombinant bacteriophage genome.
Dependencies are inferred from the claim text.