Streptococcus CRISPR-Cas9 Editing

Protocol developed from: Synefiaridou, D. & Veening, J.-W. Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V. Appl Environ Microbiol 87, e02762-20 (2021).

Reagents Needed

• pDS05 plasmid (P-80 in our freezer)
• ZnCl2-MnSO4
• CTM (Complete Transformation Medium) pH 6.8
• CTM pH 7.8
• CSP-1 peptide (in the -80 freezer; 20ul aliquots of 100uM) or CSP-2 peptide (which we do not have).

Plates Needed

• Blood TSA plates.
• Blood TSA plates with erythromycin at .25 ug/mL concentration.
• Blood TSA plates with erythromycin at .25 ug/mL concentration and 1 mM of ZnCl2-MnSO4. (Molecular weight 287.30 g/mol so 0.2873 g of ZnCl2-MnSO4).

Overall Workflow

Figure 1. Diagram of pDSO5 including where the cut site is located sgRNA, the erythromycin resistance gene ermR, and the CRISPR-cas9 genes (wtcas9). The sgRNA and the erythromycin resistance is controlled by the P3 promoter while the CRISPR-cas9 gene (wtcas9) is controlled by the zinc promoter (pZn). CRISPR-cas9 having a separate promoter controlled by the presence of zinc allows for counterselection.

1. Design sgRNA which will select what DNA fragment is being cut out of the S. pneumoniae.
2. Design and order the homologous recombinant strands (HR) which are the upstream and the downstream regions of the deletion target.
3. Generate pDSXX from pDSO5 and the sgRNA. Generate this plasmid in E. coli.
4. Transform S. pneumoniae with the pDSXX plasmid.
   1. Induce competence with the synthetic competence stimulating peptide (CSP).
   2. Add in plasmid.
   3. Select transformed colonies by plating with erythromycin (antibiotic).
5. Transform the S. pneumoniae with the pDSXX plasmid with the HR template.
   1. Induce competence with the synthetic competence stimulating peptide (CSP)
   2. Add in the HR template.
   3. Plate with zinc to induce the CRISPR system. This will mean that the only colonies that will survive will have the HR integrated into its genome.
6. Remove plasmid through using a nonpermissive temperature.

Designing sgRNA Cut Site

1. Use Benchling CRISPR guide along with the specific genome that you want to change. Find the gene what we want to disrupt, and have the CRISPR guide xxxxx
2. Design 8 primers:
   1. Two primers that, when put together, will create the proper gRNA guide RNA. There's not PCR needed for this; it is instead annealing the two primers together. Primers should be in the form XXXXXX and XXXXXX
   2. Two PCR primers that will amplify 500bp-1.5kb of the region before the cut site, which will act as a homologous recombination site. It does not need to be directly next to the cute site (and probably should not be). So, this should be upstream of gene A if we are trying to get rid of gene A.
   3. Two PCR primers that will amplify 500bp-1.5kb of the region after the cut site, which will act as a homologous recombination site. So, this should be downstream of gene A if we are trying to get rid of gene A.
   4. Two primers that will amplify the region across the cut site; ideally, we want a small PCR product if the procedure works, and a longer PCR product if the strain remains untransformed.
   5. Check with Eric about these primers and have him order them.

Talk to Eric if you are not working with a D39 derivative.

Complete Transformation Medium

• 3g Tryptic Soy Broth
• 0.1g yeast extract
• Fill up to 100ml MilliQ water and autoclave
• Add to a final concentration filter sterilized 1mM CaCl2 (found on chemical shelf), filter sterilized 0.2% BSA (Bovine Serum Albumin), and filter sterilized 1X trace mineral solution (found on chemical shelf)

Transformation Protocol with CRISPR: Adding in plasmid variant (pDSxxx)

1. Freshly grow up single colonies on a blood TSA plate of the strain to be transformed.
2. Select one colony and grow in 3ml CTM pH 6.8 at 37 degrees C until OD 0.1, which is 0.13 Absorbance
3. Preheat a microcentrifuge tube of 270ul CTM pH 7.8 to 32 degrees C using the hot block.
4. Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. Treat for 12 minutes at 37 degrees C.
   1. We use 2ul of the CSP-1 aliquot, which brings the concentration to 228ng/ml.
5. Add plasmid (pDSxxx) to 1 ug/ml final concentration — so 300ng. If this is too much DNA, it might work with half of the amount — 150ng of DNA.
   1. (final concentration of 0.7 - 2.5 ug/ml (210ng - 750ng total DNA).
6. Incubate for 20 minutes at 32 degrees C.
7. Add 30ul of grown cells (a 1:10 dilution).
8. Vortex.
9. Grow cells in CTM media at 32 degrees for 120 minutes (from step 3).
10. Plate cells on a blood TSA plate that has erythromycin at .25 ug/ml concentration in it. Use sterile cotton swab to spread the cell mixture.
11. Incubate the plate overnight in the 32 degrees C 5% CO2 incubator. (original protocol says 30 degrees C).

Transformation Protocol with CRISPR: Adding in HR template

1. Freshly grow up single colonies from the last step on a blood TSA plate of the strain to be transformed with HR template.
2. Select one colony and grow in 3ml CTM with .1 ug/mL of erythromycin pH 6.8 at 32 C until OD 0.1, which is 0.13 Absorbance
3. Preheat a microcentrifuge tube of 270 ul CTM pH 7.8 to 32 degrees C using the heat block.
4. Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. Treat for 12 minutes at 37 degrees C.
5. Add in the HR template.
6. Incubate for 20 minutes at 32 degrees C.
7. Add cells to 270 ul CTM pH 7.8 (heated in step 3).
8. Incubate for 20 minutes at 32 degrees C.
9. Plate cells on a blood TSA plate that has erythromycin at .25 ug/mL concentration and 1 mM of ZnCl2 and 1 mM MnSO4.(Based off the molecular weight of each: 0.136286 grams of ZnCL2 and 0.151 g MnSO4). Use a sterile cotton swab to spread the cell mixture.
10. Incubate at 32 degrees C.
11. Verify through sequencing or PCR that deletion has occurred.

Removing the Plasmid from the S. pneumoniae

1. Inoculate CTM and incubate at 40 degrees C.
2. Plate the liquid culture in serial dilutions to obtain single colonies.
3. Incubate overnight at 40 degrees C.
4. Screen single colonies.