Fig. 1.

Schematic representation of the strategy to test the double-strand break (DSB) activity of I-SceI expressed in T. reesei. The reporter cassette (pBJP6) with I-SceI restriction sites inserted at the cbh2 locus of a T. reesei uridine auxotrophic strain results in a uridine prototrophic strain containing two I-SceI sites surrounding the pyrG cassette (a). Heterologous expression of I-SceI is expected to generate two DSBs and the loss of the pyrG selection marker between the I-SceI sites (b). The DSB can be repaired by homologous recombination between GFP∆C and ∆NGFP adjacent to the breaks (c). After homologous recombination via the GFP direct repeats, this will result in a uridine auxotrophic strain (loss of the pyrG gene) and reconstitution of a functional GFP when correctly recombined via the direct repeat sequences (d). The resulting strain can be either screened for uridine auxotrophy or/and for GFP expression to monitor I-SceI activity