Meganuclease I-SceI based system for gene deletions

From ActinoBase

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.

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:

Workflow Overview

  • Design primers to amplify both flanking regions
  • Clone both flanking regions into pIJ12738
  • Transform vector into strain to be mutated
  • Select for successful transformants
  • Transform successful transformant with pIJ12742
  • Select for successful deletion mutants
  • Remove pIJ12742 from your mutant strain

Designing primers to amplify flanking regions

Identify the genome sequence which you would like to delete, whether an individual gene or an entire biosynthetic gene cluster.

To amplify the left flanking region:

The reverse primer must match the 18-22 bp immediately adjacent to the region to be deleted so you do not have flexibility on its design. Now find a suitable forward primer which is located 1.2-2.5 Kb upstream of that reverse primer. Make sure you add suitable restriction enzyme sites present in the MCS of pIJ12738 to your primer sequences and that the enzymes chosen do not cut the amplified left flanking region.


To amplify the right flanking region:

The forward primer must match the 18-22 bp immediately adjacent to the region to be deleted so you do not have flexibility on its design. Now find a suitable reverse primer which is located 1.2-2.5 Kb downstream of that forward primer. Make sure you add suitable restriction enzyme sites present in the MCS of pIJ12738 to your primer sequences and that the enzymes chosen do not cut the amplified right flanking region.

Cloning both flanking regions into pIJ12738

Amplify the left and right flanking regions. You might have to optimise the PCR conditions using DMSO and a temperature gradient, see High GC PCR.

Clone the left and right flanking regions into pIJ12738 (either sequentially or simultaneously). Check that your plasmid contains the desired flanking regions.

Transform the plasmid containing the left and right flanking regions into the strain of interest

Transfer the pIJ12738 derivative generated above into the actinomycete strain of interest using an optimised transformation/conjugation protocol suitable for that particular strain and using apramycin for selection. If it is a newly isolated strain, optimise the transformation /conjugation efficiency using pIJ12742. This can be done at the same time as you are cloning the flanking regions into pIJ12738 to save time.

Select for successful transformants

Apramycin resistant transformants (final apramycin concentration is strain dependent) should be replica plated onto a fresh apramycin containing plate. You can use colony PCR to check for the presence of the plasmid (see general guide on the High GC PCR page). If colony PCR does not work in your strain, the extract genomic DNA and check that your pIJ12738 derivative has been successfully integrated in the genome.

Once you have confirmed a successful transformant, obtain a spore/mycelia/gycerol stock (use the method which you would normally use to preserve your strain) of the above transformant.

Transform your transformant with pIJ12742

Transfer the I-sceI delivery vector pIJ12742 (I-sceI under the control of the constitutive strong promoter ermE*p) into the transformant strain obtained in step 1.6 using an optimised transformation/conjugation protocol suitable for that particular strain and using thiostrepton for selection.

Select successful deletion mutants in your strain of interest

Thiostrepton resistant transformants (final thiostrepton concentration is strain dependent) should be replica plated onto a fresh thiostrepton containing plate. You can use colony PCR to check if your transformant has the wild-type or deletion mutant genotype. If colony PCR does not work in your strain, the extract genomic DNA and check if your transformant has the wild-type or deletion mutant genotype.

Once you have confirmed a successful mutant genotype, obtain a spore/mycelia/gycerol stock (use the method which you would normally use to preserve your strain) of the above deletion mutant and get ready to do some cool experiments with your mutant!

Remove pIJ12742 from your deletion mutant strain

pIJ12742 contains a temperature sensitive replicon and therefore loss of this plasmid can be easily achieved by growing the strain at 37ºC in the absence of thiostrepton, leaving a clean deletion mutant strain (no antibiotic resistance markers) which can undergo further genetic manipulation if required. For strains which are unable to grow at such temperature, two or three rounds of growth at 30ºC in the absence of thiostrepton should also promote loss of pIJ12742.

Protocol graphical overview and plasmids maps

Overview of the protocol developed to obtain markerless gene deletions in actinomycetes. pIJ12738 containing the I-SceI recognition sequence (shown by a pink circle) and the flanking regions (left and right) of the chromosomal region to be deleted is transformed into the strain of interest. Apramycin resistant transformants are analysed by PCR to confirm integration of the plasmid. A single exconjugant is selected as recipient of the delivery vector pIJ12742 expressing the I-SceI meganuclease gene under ermE*p. After transformation, transformants are selected with thiostrepton. I-SceI creates double strand breaks at its introduced recognition sequence, and the only genomes to survive are those that undergo homologous recombination to reconstitute an intact chromosome. If recombination occurs in the same flanking region that was used in the first cross-over event, then the exconjugant will revert to a wild type genotype. If recombination takes place in the other flanking region, the exconjugant will have a mutant genotype. Transformants are analysed by PCR to confirm the required mutant genotype. To ensure loss of the I-SceI delivery vector, the mutant strain is grown at 37 0C, and loss of the vector confirmed by PCR.


Restriction maps of the plasmids constructed to make markerless deletion mutants in any actinomycete strain of interest. aac(3)IV, apramycin resistance gene; ori, E. coli origin of replication; oriT, origin of transfer; MCS, multiple cloning site; lacZ, α fragment of lacZ; rep, origin of replication from pSG5;tsr, thiostrepton resistance gene; fd, transcriptional terminator from phage fd; to, transcriptional terminator; ermE*p, mutated constitutive promoter from ermE*.




























































References

For further information about this deletion system, how to obtain the plasmids and for citation of this protocol:

  • Fernández-Martínez LT & Bibb MJ. Use of the Meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes. Sci. Rep.4, 7100; DOI:10.1038/srep07100 (2014).


Protocol adapted for ActinoBase by Dr. Lorena Fernández-Martínez, a group leader at Edge Hill University