Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies

Abstract

Site-specific recombinases (SSRs) such as Cre are widely used in gene targeting and genetic approaches for cell labeling and manipulation. They mediate DNA strand exchange between two DNA molecules at dedicated recognition sites. Precise understanding of the Cre recombination mechanism, including the role of individual base pairs in its loxP target site, guided the generation of mutant lox sites that specifically recombine with themselves but not with the wild type loxP. This has led to the development of a variety of combinatorial Cre-dependent genetic strategies, such as multicolor reporters, irreversible inversions, or recombination-mediated cassette exchange. Dre, a Cre-related phage integrase that recognizes roxP sites, does not cross-react with the Cre-loxP system, but has similar recombination efficiency. We have previously described intersectional genetic strategies combining Dre and Cre. We now report a mutagenesis screen aimed at identifying roxP base pairs critical for self-recognition. We describe several rox variant sites that are incompatible with roxP, but are able to efficiently recombine with themselves in either purified systems or bacterial and eukaryotic tissue culture systems. These newly identified rox sites are not recognized by Cre, thus enabling potential combinatorial strategies involving Cre, Dre, and target loci including multiple loxP and roxP variants.

Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods

“…A number of Cre orthologues, including Dre and VCre, have recently been identified by genome mining and demonstrated to have in vivo function 23 – 25 . Additionally, Chuang et al 26 carried out an extensive mutant library screening, and identified novel rox sites, which can efficiently recombine with themselves in vivo but have minimal to no reactivity with wild-type (WT) rox sites 26 .…”

Evidence of a developmental origin of beta-cell heterogeneity using a dual lineage tracing technology

“…Importantly, Dre does not crossreact with the Cre/LoxP system, but has similar recombination efficiency (Sauer and McDermott, 2004). The Dre/RoxP system has been previously tested in some settings (Chuang et al, 2015), but not yet widely used in gene targeting studies to generate lineage manipulation in animals.…”

A knock‐in allele of En1 expressing dre recombinase

“…To specifically target cells that express En1 in the adult, without interference from embryonic En1 domains, En1 Dre can be used in conjunction with injection of Dre‐responsive and Dre/Cre‐responsive viral constructs encoding indicators and effectors (Chuang et al ., ; Fenno et al ., ; Madisen et al ., ). Several of these recently described constructs utilize engineered mutant rox target sites to permit control of indicator or effector gene expression by Dre‐dependent inversion and excision similar to Cre‐dependent FLEx/DIO constructs (Chuang et al ., ; Fenno et al ., ). Given these possibilities, we expect that En1 Dre will become an important part of the genetic toolbox for analysis of neurons in the mammalian midbrain and hindbrain.…”

“…Thus, eGFP will be expressed in En1 Dre ; RC::RLTG brain only if Dre recombines the loxP sites as well as the rox sites. We observed no eGFP labeled cells ( n = 4 brains), consistent with prior observations that Dre and loxP sites are generally incompatible in transgenic systems (Chuang et al ., ; Park and Leach, ; Plummer et al ., ), with significant cross‐reactivity only observed in the context of viral vector‐driven overexpression (Fenno et al ., ). These results confirm that En1 Dre is suitable for use in intersectional genetic analyses together with Cre driver alleles.…”