DNA methylation occurring in Cre-expressing cells inhibits loxP recombination and silences loxP-sandwiched genes.

Abstract

● The low DNA recombination efficiency of site-specific recombinase systems in plants limits their application; however, the underlying mechanism is unknown. ● We evaluate the gene deletion performance of four recombinase systems (Cre/loxP, Flp/FRT, KD/KDRT, and B3/B3RT) in tobacco where the recombinases are under the control of germline-specific promoters. ● We find that the expression of these recombinases results mostly in gene silencing rather than gene deletion. Using the Cre/loxP system as a model, we reveal that the region flanked by loxP sites (floxed) is hypermethylated, which prevents floxed genes from deletion while silences the expression of the genes. We further show CG methylation alone in recombinase binding element of loxP site is unable to impede gene deletion; instead, CHH methylation in the crossover region is required to inhibit loxP recombination. ● Our study illustrates the important role of recombinase-induced DNA methylation in the inhibition of site-specific DNA recombination and uncovers the mechanism underlying the recombinase-associated gene silence in plants.