Improved Lox SItes for Cre Recombination Plants

Provides recombination sites for the most widely used recombinase that facilitate increased plant activity

Background
Site-specific recombinases are widely used molecular tools for performing DNA manipulations both in vitro and in vivo, with applications in research and biotechnology. This includes deletion, integration, and inversion of target sequences of interest. Cre acts upon recognition sequences which are called lox sites, the most widely used of which is loxP, which is naturally present in the phage P1 genome. The inventors developed novel lox sequences, and a method for generating them, which are designed to increase activity in plant cells. Because plants have different methods and contexts of DNA methylation compared with animals, cytosine methylation prevents efficient recombination of target sites (Figure 1). Discovery and implementation of lox sites which are resistant to cytosine methylation improves their activity in plants.

_{Figure 1: Steric hinderance by methylated cytosines impairs recombination by Cre, especially in plant somatic cells.}

Technology Description
To discover lox sites which lack cytosines in key positions, the inventors developed a novel screening method. Resulting lox sites from this screen with cytosine-free sequences are tested for activity through cloning two direct repeats in plasmids and treating with purified Cre recombinase to generate excision products. To implement these lox sequences and test for increased recombination efficiency in plants, well-performing sites are cloned in direct repeats, the first placed in the 5’ UTR of a fluorescent reporter gene, and the second placed after the terminator of the same fluorescent gene. A second fluorescent reporter is placed downstream of this site without a promoter such that excision via Cre of the first product turns on expression of the second reporter (Figure 2). Testing of these vectors with a constitutively expressed Cre in tobacco agroinfiltration assays shows significantly increased transgene excision relative to the same vector with traditional loxP sites. The same result was found in stable transgenics in the hybrid poplar P. tremula x alba (Figure 3).

_{Figure 2: Vectors for testing recombination rates of improved lox sites in plants (top panel). When used in tobacco agroinfiltration assays, successful excision was obtained (GFP) with cytosine-attenuated loxP sequences but not with traditional loxP sequences (bottom panel).}

_{Figure 3: In Populus tremula x alba stable transgenic plants expressing fluorescent switch vectors (that show recombinase activity and its lack with different fluorescent reporter genes). Plants with cytosine-attenuated loxP recombined, as shown by the green eGFP bars (C). Without the technology, there were no excised shoots recovered (B). Presence of Cre decreased transgenic plant recovery relative to a no-cre control, but only to a modest degree that would still allow productive use of the technology (A vs. B and C).}

Features & Benefits

Provides recombination sites for the most widely used recombinase which facilitate increased plant activity by making them resistant to cytosine methylation which occurs in unique contexts in plants

Applications