One-step recombination-mediated cassette exchange (RMCE) in Drosophila suzukii

Pratima Chennuri, Ph.D. is a Postdoctoral Research Associate in the Department of Entomology at Texas A&M University and is interested in Gene Drives, DNA Repair, small RNA pathways (piRNAs) and transposons. MORE ABOUT THE AUTHOR

Schetelig et al. (2018) describe a relatively efficient recombinase-mediated cassette exchange (RMCE) system for engineering the recently sequenced genome (Chiu et al., 2013) of the spotted wing drosophila, Drosophila suzukii, which is a significant pest of small and stone fruits.

RMCE is a method of creating landing sites in an organism’s genome that are amenable to further engineering through exchange, deletion or addition of new segments of DNA. These RMCE landing sites contain short variable DNA sequences (lox or FRT) that are recognized and recombined by specific recombinases, such as Cre (lox sites) or Flippase (FRT sites).

Adult male spotted wing drosophila, Drosophilia suzukii (Matsumura). Photograph by Martin Hauser, California Department of Food and Agriculture.

Introduction of heterospecific recombination sites, such as loxN and lox2272, as employed by the authors of this paper, can be used as a RMCE template for replacing or exchanging the intervening DNA sequence sandwiched between the two heterospecific sites. This exchange can be achieved by providing a donor DNA sequence carrying the same heterospecific sites sandwiching a DNA cargo of interest accompanied by a fluorescent reporter marking the exchange, followed by molecular confirmation via diagnostic junctional PCRs.

The basic concept of Recombination Cassette Mediated Exchange illustrated with the Flp/FRT system but other recombinase systems such as the Cre/LOX system can be used to accomplish the same thing. Image credit via license: CC BY 4.0

The authors used transposon-mediated transgenesis in D. suzukii to create lines containing landing sites marked by EGFP with loxN/lox2272 heterospecific sites sandwiching CFP (CYGFP). They then co-injected 82 eggs from one of the lines with a donor DNA construct carrying the same heterospecific sites sandwiching DsRed with a source of Cre recombinase, and scored for transformants where CFP was replaced by DsRed. This produced two G0 lines and for each of these lines, they report a relatively efficient RMCE frequency of 20%.

They refer to similar studies in Drosophila melanogaster (Horn and Handler, 2005) and Aedes aegypti (Hacker et al., 2017) and compare the efficiency of exchange events where both heterospecific sites were recombined vs single-site recombination events, and find that with D. melanogaster, nearly 50% of the events were single-site while all the initial events in A. aegypti were single-site.

Variants of LoxP sites, shown as colored triangles, all are substrates for Cre recombinase but only involving identical sites. Heteroallelic combinations will not recombine. mage credit via license: CC BY 4.0

While acknowledging that their sample size was rather small, the authors indicate that one-step RMCE, i.e RMCE achieved in a single generation was much more efficient in Drosophila than Aedes, where secondary single recombination events in subsequent generations were required for complete RMCE. One needs to, however, bear in mind that these observations are subject to position effects and the context of the genomic landscape and as suggested by the authors, further evaluation of the reported relative RMCE efficiencies is required.

The application of CRISPR/Cas9-mediated genome engineering for creating landing sites that are non-random with rigorous evaluation of off-target effects are important future directions for developing effective targeted biological control of arthropod pests.

Schetelig, M.F., Yan, Y., Handler, A. M. (2018) Genomic targeting by recombinase-mediated cassette exchange in the spotted wing drosophila, Drosophila suzukii. Insect Mol Biol. doi: 10.1111/imb.12537.




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