Methods of unidirectional, site-specific integration into a genome, compositions and kits for practicing the same
View Patent ↗The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided system includes a site-specific integrating expression cassette (INTEC) vector, consisting of (a) a polynucleotide of interest operably linked to a promoter, (b) a single recombination site, and (c) a hybrid recombination site. In using the subject systems for site-specific integration, the INTEC vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific recombinase. Also provided are kits that include the subject systems. The subjects systems, methods and kits find use in a variety of different applications, several representative ones of which are described in detail as well.
1. A site-specific integrating expression cassette (INTEC) vector, wherein the INTEC vector consists of:
(a) a polynucleotide of interest operably linked to a promoter,
(b) a single recombination site selected from an attP site, an attB site, a pseudo attP site, and a pseudo attB site that is a substrate for recombination by a unidirectional site-specific recombinase selected from a φC31 phage recombinase, a TP901-1 phage recombinase, or an R4 phage recombinase, wherein said single recombination site is a substrate for recombination with a recombination site in the genome of a target cell by said unidirectional site-specific recombinase, and
(c) a hybrid recombination site selected from an attL site and an attR site, wherein the hybrid recombination site is not a substrate for said unidirectional site-specific recombinase.
2. The INTEC vector of claim 1 , wherein said single recombination site is an attP site or an attB site.
3. The INTEC vector of claim 1 , wherein said single recombination site is a pseudo attP site or a pseudo attB site.
4. The INTEC vector of claim 1 , wherein said unidirectional site-specific recombinase is φC31 phage recombinase.
5. The INTEC vector of claim 1 , wherein said polynucleotide of interest comprises a coding sequence.