Feb 27, 2023

Public workspaceCRISPR/Cas9 in vitro assembled gene deletion protocol for Coccidioides posadasii

DOI
dx.doi.org/10.17504/protocols.io.q26g7y663gwz/v1
  • 1Northern Arizona University;
  • 2Arizona State University
  • Bridget M. Barker: Lab PI;
Bridget M. Barker
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DOI: dx.doi.org/10.17504/protocols.io.q26g7y663gwz/v1
Protocol Citation: Bridget M. Barker, Ashley Itogawa, Heather Mead, Austin Blackmon, mitchellbryant 2023. CRISPR/Cas9 in vitro assembled gene deletion protocol for Coccidioides posadasii. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y663gwz/v1
Manuscript citation:

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 23, 2023
Last Modified: May 25, 2023
Protocol Integer ID: 77531
Keywords: CRISPR, Cas9, Coccidioides
Funders Acknowledgement:
NIH/NIAID
Grant ID: 5U19AI166798-02
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Abstract
This protocol was developed to use CRISPR/Cas9 technology to delete genes in Coccidioides posadasii. This methodology is useful for creating targeted deletions in any strain of Coccidioides, and has been successful in our lab. The method will allow for users to design and implement targets of their preference, and specific details regarding resistance markers and targets was removed. The user will need to define these specific parameters. This protocol will not work if a gene is essential, and certain genomic regions may be recalcitrant to modification.
Materials
  1. MOPS buffer containing sorbitol (MS)
A.10 mM MOPS, 1 M sorbitol (pH 6.5)
B.500 ml/transformation experiment

2. MSC buffer (MSC)
A.MS with20 mM CaCl2
B.100 ml/transformation experiment

3. 0.9% phosphate buffered saline (PBS)

4. DNA repair template (hphor other selective marker as appropriate)

5. Cas9:crRNA:tracrRNA ribonucleoprotein (RNP) complex (See Table 1)

6. 60% (w/v) polyethylene glycol (PEG)
A.Made in MSC
B.PEG quality varies depending on the source. The precise function of PEG is not known, although it may contribute to the efficiency of DNA construct uptake by protoplasts (2). Long term storage of PEG solution will decrease transformation efficiency. Do not use PEG solution >3 months old.

7. GYES soft agar
A.1% glucose, 0.5% yeast extract, 1 M sucrose, 0.7% Bacto agar
B.25 ml/transformation experiment

8. GYES agar plates
A.1% glucose, 0.5% yeast extract, 1 M sucrose, 2% Bacto agar

9. 1X GYE soft agar with appropriate concentration of a selective agent
A.1% glucose, 0.5% yeast extract, 0.7% Bacto agar
B.Potential selective agents
i.e. 375 µl hygromycin B (Hyg) (Sigma)/L media

10. 1X GYE agar plates with appropriate concentration of a selective agent
A.1% glucose, 0.5% yeast extract, 2% Bacto agar
B.Potential selective agents
i.e. 375 µl hygromycin B (Hyg) (Sigma)/L media
C.May be made in untreated 6-well culture plates or small petri plates for initial selection step. Standard size petri plates (150 mm) are best for subsequent purification steps.







Safety warnings
1. The selective media used for isolating Coccidioides spp. transformants includes an acutely toxic compound: hygromycin B. No specific risk mitigation is required for this compound due to the low amount and dispersed nature of this compound in the semi-solid/solid waste generated in this SOP. However, concentrated hygromycin B stocks used to prepare media should be handled in a fume hood and all appropriate PPE should be worn (i.e., lab coat, gloves, eye protection). If a spill involving this compound occurs, spill mitigation should include removing any potentially contaminated PPE and a thorough hand washing.
Some of the buffer reagents (e.g., MOPS) contain compounds that are incompatible with bleach. No specific risk mitigation is required for these reagents due to the diluted nature of these reagents in the waste generated in this SOP
Before start
Read materials page and order and make needed reagents prior to start. The creation of protoplasts should be complete before starting this procedure
Prepare all needed reagents, prepare RNP complex, prepare protoplasts on ice, warm GYES agar plates to 30C, warm 60% PEG to room temperature: see materials list for recipes and ordering information.
2d
Place 1.5 ml microcentrifuge tube(s) containing protoplasts and other supplies needed in BSC.
A.Keep protoplasts on cold beads/ice.
B.If the protoplasts were stored at -80°C, they will need to thaw on cold beads.
3m
Adjust protoplast volume in each 1.5 ml microcentrifuge tube to be processed to ~100 µl. Use additional 1.5 microcentrifuge tubes as necessary.
A.One 1.5 ml microcentrifuge tube containing ~100 µl protoplasts is needed per transformation.
B. Avoid multiple freeze thaws of protoplasts
5m
Add desired DNA repair template (1 - 3 µg) to each 1.5 ml microcentrifuge tube containing ~100 µl protoplasts and gently stir with pipette tip to mix; do not pipette or vortex.
A.DNA repair template volume should be ~10 µl and no more than 20 µl.
B.DNA repair template concentration must be at least 1 µg.
1m
Add 26.5 µl RNP complex to each protoplast:DNA mixture in a 1.5 ml microcentrifuge tube.Do not mix.
A.Cas9 endonuclease concentration should be maximized.
1m
Add 25 µl 60% PEG to each protoplast:DNA:RNP complex mixture in a 1.5 ml microcentrifuge tube and mix by slowly pipetting twice
2m
Incubate 1.5 ml microcentrifuge tubes on cold beads for 50 min.
50m
Add 900 µl additional 60% PEG to each 1.5 ml microcentrifuge tube and mix gently by inversion until 60% PEG is fully incorporated.
2m
Lay 1.5 ml microcentrifuge tubes horizontally on a paper towel on the BSC work surface and incubate at room temperature for 30 min
30m
Using a p1000 with large pore tip, overlay each suspension onto a pre-warmed GYES agar plate and gently rotate or use soft loop to spread suspension evenly
5m
Place GYES plates agar side down in incubator at 30°C for 48 hr to allow cell wall regeneration and colony growth.
Note: At this point, the protoplasts are delicate due to a lack of cell walls; the cell walls should regenerate in 12 - 24 h
Observe for growth, should see faint growth by 48 hours. Occasionally up to 72 hours is needed.
2d
When growth is observed:
Prepare 1X GYE soft agar with appropriate selective agent
i. Melt 1X GYE soft agar by microwaving.
ii. Transfer needed amount 1X GYE soft agar to 50 ml conical centrifuge tube and incubate at 46°C.
iii. Add appropriate amount selective agent to melted 1X GYE soft agar.
NOTE: Many selective agents are temperature sensitive. Ensure 1X GYE soft agar has cooled to 46°C (or appropriate temperature for specific selective agent) before adding selective agent
30m
Add 2.5 mL 1xGYE soft agar with selective agent to each GYES plate with emerging fungal growth and incubate agar side down at 30C for 4-7 days. Avoid light is selective agent is light sensitive.
1w
When colonies are observed (small, star shaped) growing through the selective media, pick colonies and transfer to fresh 1xGYE agar in culture plates (12-well or 6-well depending on preference) and incubate at 30C for 7-14 days, until colonies reach ~2cm in diameter.
2w
Re-plate surviving colonies in the same manner as step 14, and incubate at 30C for 7 days, until colonies reach ~2cm in diameter
1w
Select a well grown, isolated colony and mark on the back of the plate. Sample the outer edge of the selected colony using an inoculating loop and use to streak for isolated colonies on a fresh selective agar plate. Repeat steps for desired number of transformants (we usually select 12). Incubate selective agar plates at 30°C for 3-5 days
1w
Pick single colonies and transfer to fresh selective media plates. Passage 1-2 times to ensure homokaryons. From final plate, scrape a small amount of mycelia to a DNA lysis buffer and extract DNA using appropriate method.
2w
Screen extracted DNA for homokaryotic transformants using PCR, design PCR primers to target both selective marker and DNA segment that should be removed in transformant
1d
Southern blot analysis with a gene-specific probe on PCR selected transformants can be used to confirm the formation and purification of desired homokaryotic mutants
Protocol references
Al Abdallah, Q., W. Ge, and J. R. Fortwendel. 2017. A simple and universal system for gene manipulation inAspergillusfumigatus. In vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates. mSphere. Nov 22;2(6):e00446-17. doi: 10.1128/mSphere.00446-17. PMID: 29202040; PMCID: PMC5700375.