Meganuclease I-SceI based system for gene deletions

Gene deletion in actinomycetes using I-SceI meganuclease

This protocol allows the reader to make clean deletions in any actinomycete species using an efficient method to generate gene or gene cluster deletions by forcing homologous recombination using the meganuclase SceI. SceI forces double strand breaks on it’s 18 bp recognition sequence forcing the cell to undergo homologous recombination in order to survive. This method does not introduce any other changes to the targeted organism’s genome, making sequential deletions easy to carry out. Also, the only information that you require prior to deletion of the region of interest is the sequence of the flanking regions (about 1.2 to 2.5 Kb either side of the region that you want to delete). This method can be used on any actinomycete strain whose genome lacks the SceI 18 bp recognition sequence TAGGGATAACAGGGTAAT.

This genome editing tool is a two-step protocol in which first the flanking regions are amplified and cloned into pIJ12738 and the resulting vector is conjugated into the target organism. Once the pIJ12738 derivative is integrated in the actinomycete’s chromosome, a vector carrying a codon optimised I-sceI gene for expression in actinomycetes (pIJ12742) is conjugated into the above strain. I-sceI expression produces a double strand chromosomal break at a unique introduced 18 base pair I-SceI recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype.

1.1 Organisms

This protocol should work on any actinomycete species whose genome lacks the SceI recognition site (see above). So far this protocol has been confirmed to work for the following actinomycete strains:

• Streptomyces coelicolor
• Streptomyces venezuelae
• Streptomyces clavurigerus
• Microbispora corallina
• Rhodococcus sp.