Recombinase technology: applications and possibilities

Abstract

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes.

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy

“…For the cotransformation, site-specific recombination, and other T-DNA-based strategies for marker gene removal (Ebinuma et al, 2001;Wang et al, 2011;Kapusi et al, 2013;Yau and Stewart, 2013), the complex transgene integration patterns such as multiple T-DNA loci, tandem-linked T-DNA copies, T-DNA truncations, and cotransferred non-T-DNA vector backbone sequence in transgenic plants (Kim et al, 2003;Rai et al, 2007) may complicate the molecular analysis of transgene integration and affect the stability of transgene expression. In this study, we validated that the maize Ac/Ds transposable element can be routinely used to generate marker-free transgenic plants in rice.…”

“…Several strategies have been developed to remove selectable markers from transgenic plants while retaining the gene of interest (GOI; Ebinuma et al, 2001;Cotsaftis et al, 2002;Wang et al, 2011;Kapusi et al, 2013;Yau and Stewart, 2013;Oliva et al, 2014). Transposition is an advantageous strategy for marker gene removal, because it allows intact transgene insertion with defined boundaries and requires only a few primary transformants (Cotsaftis et al, 2002).…”

Next generation biotechnology: how sophisticated constructs lead to further insights and new approaches towards biotechnology’s demands

“…These enzymes are able to either invert or excise sequences between recognition sites as part of an intramolecular recombination, but also to perform an intermolecular site-directed integration of DNA, depending on the orientation and type of recognition sites. These properties have been reported in several plants (Lyznik et al 1993 -for an overview, see Wang & Yau 2011) and are also wildly known in the field of genomic engineering with mammalian and drosophila cells as well as several prokaryotes (Kolb 2002). General considerations for the use of recombinases and the use of variable constructs should be the right choice of promoters, regulating when or where which recombinase should be active (Van Ex et al 2009).…”

Development of a Simple and Efficient System for Excising Selectable Markers in Arabidopsis Using a Minimal Promoter:: Cre Fusion Construct

“…In the homologous recombination strategy, large deletions via illegitimate recombination are often observed and the efficiency of recovering selectable marker-free transgenic plants is low (Zubko et al, 2000). Using the site-specific recombinase-mediated excision systems, such as Cre/loxP and Flp/frt, the recombinases Cre and Flp act on their target sequences, loxP and frt, respectively, that flank selectable marker genes and excise them from the transgenic plants (Abremski and Hoess, 1984;Miki and McHugh, 2004;Ow, 2002;Wang et al, 2010). Among these strategies, the site-specific recombination technology using Cre/loxP has been widely tested to create transgenic plants without retention of selectable marker genes.…”