Streptococcus CRISPR-Cas9 Editing: Difference between revisions
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PreDec2022>EricMiller (Created page with " == Reagents Needed== * pSD05 plasmid (P-80 in our freezer) == Designing gRNA Cut Site== # 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 # Design 8 primers: ## 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 XXXX...") |
PreDec2022>MSmith9 (Updating this protocol to be more specific for the CRISPR- Cas 9 system and adding in HR. Additionally, adding a workflow that shows how the multiple parts work together.) |
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Line 1: | Line 1: | ||
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== | == 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 == | |||
== Designing | # Design sgRNA which will select what DNA fragment is being cut out of the S. pneumoniae. | ||
# Design and order the homologous recombinant strands (HR) which are the upstream and the downstream regions of the deletion target. | |||
# Generate pDSXX from pDSO5 and the sgRNA. Generate this plasmid in E. coli. | |||
# Transform S. pneumoniae with the pDSXX plasmid. | |||
## Induce competence with the synthetic competence stimulating peptide (CSP). | |||
## Add in plasmid. | |||
## Select transformed colonies by plating with erythromycin (antibiotic). | |||
# Transform the S. pneumoniae with the pDSXX plasmid with the HR template. | |||
## Induce competence with the synthetic competence stimulating peptide (CSP) | |||
## Add in the HR template. | |||
## 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. | |||
# Remove plasmid through using a nonpermissive temperature. | |||
== Designing sgRNA Cut Site == | |||
# 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 | # 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 | ||
# Design 8 primers: | # Design 8 primers: | ||
Line 14: | Line 38: | ||
## Check with Eric about these primers and have him order them. | ## 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 == | == Complete Transformation Medium == | ||
Line 27: | Line 46: | ||
* 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) | * 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)== | ||
# Freshly grow up single colonies on a blood TSA plate of the strain to be transformed. | # Freshly grow up single colonies on a blood TSA plate of the strain to be transformed. | ||
# Select one colony and grow in 3ml CTM pH 6.8 until OD 0.3, which is 0.39 Absorbance | # Select one colony and grow in 3ml CTM pH 6.8 at 37 degrees C until OD 0.3, which is 0.39 Absorbance | ||
# Preheat a microcentrifuge tube of 270ul CTM pH 7.8 to | # Preheat a microcentrifuge tube of 270ul CTM pH 7.8 to 30 degrees C using the hot block. | ||
# 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. | # Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. Treat for 12 minutes at 37 degrees C. | ||
# Add | ## We use 2ul of the CSP-1 aliquot, which brings the concentration to 228ng/ml. | ||
# 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. | |||
## (final concentration of 0.7 - 2.5 ug/ml (210ng - 750ng total DNA). | |||
# Incubate for 20 minutes at 30 degrees C. | |||
# Add 30ul of grown cells (a 1:10 dilution). | # Add 30ul of grown cells (a 1:10 dilution). | ||
# Vortex | # Vortex. | ||
# | # Grow cells in CTM media at 30 degrees for 120 minutes (from step 3). | ||
# Plate cells on a blood TSA plate that has | # 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. | ||
# Incubate the plate overnight in the | # Incubate the plate overnight in the 30 degrees C 5% CO2 incubator. (original protocol says 30 degrees C). | ||
== Transformation Protocol with CRISPR: Adding in HR template == | |||
# Freshly grow up single colonies from the last step on a blood TSA plate of the strain to be transformed with HR template. | |||
# Select one colony and grow in 3ml CTM with .1 ug/mL of erythromycin pH 6.8 at 30 C until OD 0.3, which is 0.39 Absorbance | |||
# Preheat a microcentrifuge tube of 270 ul CTM pH 7.8 to 30 degrees C using the heat block. | |||
# Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. Treat for 12 minutes at 37 degrees C. | |||
# Add in the HR template. | |||
# Incubate for 20 minutes at 30 degrees C. | |||
# Add cells to 270ul CTM pH 7.8 (heated in step 3). | |||
# Incubate for 20 minutes at 30 degrees C. | |||
# Plate cells on a blood TSA plate that has 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). Use a sterile cotton swab to spread the cell mixture. | |||
# Incubate at 30 C. | |||
# Verify through sequencing or PCR that deletion has occurred. | |||
== | == Removing the Plasmid from the S. pneumoniae == | ||
# Inoculate CTM and incubate at 40 degrees C. | |||
# Plate the liquid culture in serial dilutions to obtain single colonies. | |||
# Incubate overnight at 40 degrees C. | |||
# Screen single colonies. |
Revision as of 21:52, 31 August 2022
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
- Design sgRNA which will select what DNA fragment is being cut out of the S. pneumoniae.
- Design and order the homologous recombinant strands (HR) which are the upstream and the downstream regions of the deletion target.
- Generate pDSXX from pDSO5 and the sgRNA. Generate this plasmid in E. coli.
- Transform S. pneumoniae with the pDSXX plasmid.
- Induce competence with the synthetic competence stimulating peptide (CSP).
- Add in plasmid.
- Select transformed colonies by plating with erythromycin (antibiotic).
- Transform the S. pneumoniae with the pDSXX plasmid with the HR template.
- Induce competence with the synthetic competence stimulating peptide (CSP)
- Add in the HR template.
- 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.
- Remove plasmid through using a nonpermissive temperature.
Designing sgRNA Cut Site
- 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
- Design 8 primers:
- 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
- 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.
- 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.
- 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.
- 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)
- Freshly grow up single colonies on a blood TSA plate of the strain to be transformed.
- Select one colony and grow in 3ml CTM pH 6.8 at 37 degrees C until OD 0.3, which is 0.39 Absorbance
- Preheat a microcentrifuge tube of 270ul CTM pH 7.8 to 30 degrees C using the hot block.
- Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. Treat for 12 minutes at 37 degrees C.
- We use 2ul of the CSP-1 aliquot, which brings the concentration to 228ng/ml.
- 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.
- (final concentration of 0.7 - 2.5 ug/ml (210ng - 750ng total DNA).
- Incubate for 20 minutes at 30 degrees C.
- Add 30ul of grown cells (a 1:10 dilution).
- Vortex.
- Grow cells in CTM media at 30 degrees for 120 minutes (from step 3).
- 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.
- Incubate the plate overnight in the 30 degrees C 5% CO2 incubator. (original protocol says 30 degrees C).
Transformation Protocol with CRISPR: Adding in HR template
- Freshly grow up single colonies from the last step on a blood TSA plate of the strain to be transformed with HR template.
- Select one colony and grow in 3ml CTM with .1 ug/mL of erythromycin pH 6.8 at 30 C until OD 0.3, which is 0.39 Absorbance
- Preheat a microcentrifuge tube of 270 ul CTM pH 7.8 to 30 degrees C using the heat block.
- Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. Treat for 12 minutes at 37 degrees C.
- Add in the HR template.
- Incubate for 20 minutes at 30 degrees C.
- Add cells to 270ul CTM pH 7.8 (heated in step 3).
- Incubate for 20 minutes at 30 degrees C.
- Plate cells on a blood TSA plate that has 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). Use a sterile cotton swab to spread the cell mixture.
- Incubate at 30 C.
- Verify through sequencing or PCR that deletion has occurred.
Removing the Plasmid from the S. pneumoniae
- Inoculate CTM and incubate at 40 degrees C.
- Plate the liquid culture in serial dilutions to obtain single colonies.
- Incubate overnight at 40 degrees C.
- Screen single colonies.