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Reagents Needed

• pSD05 plasmid (P-80 in our freezer)

Designing gRNA 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.

• 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). 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)

Competence Protocol

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 until OD 0.3, which is 0.39 Absorbance
3. Preheat a microcentrifuge tube of 270ul CTM pH 7.8 to 37 degrees using the hot block.
4. Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. We use 2ul of the CSP-1 aliquot, which brings the concentration to 228ng/ml.
5. Add DNA 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.
6. Add 30ul of grown cells (a 1:10 dilution).
7. Vortex
8. Incubate at 37 degrees using the hot block for 60 minutes.
9. Plate cells on a blood TSA plate that has appropriate antibiotics in it. Use sterile cotton swab to spread the cell mixture.
10. Incubate the plate overnight in the 37 degrees C 5% CO2 incubator.

Competence Protocol with CRISPR

• As above, but add the editing construct at a final concentration of 0.7 - 2.5 ug/ml (210ng - 750ng total DNA)
• Incubate at 37 degrees using the hot block for 20 minutes.
• Add the CRISPR targeting construct at a final concentration of 0.7 - 2.5 ug/ml (mirroring the first set of DNA), and vortex.
• Incubate at 37 degrees using the hot block for 40 minutes.

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