TITLE: CRISPR/Cas9 in vitro assembled gene deletion protocol for Coccidioides posadasii
AUTHORS: Bridget M. Barker, Ashley Itogawa, Heather Mead, Austin Blackmon, mitchellbryant

[DESCRIPTION]
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.

[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

[STEPS]
1. 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.
2. 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.
3. 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
4. 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.
5. 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.
6. 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
7. Incubate 1.5 ml microcentrifuge tubes on cold beads for 50 min.
8. Add 900 µl additional 60% PEG to each 1.5 ml microcentrifuge tube and mix gently by inversion until 60% PEG is fully incorporated.
9. Lay 1.5 ml microcentrifuge tubes horizontally on a paper towel on the BSC work surface and incubate at room temperature for 30 min
10. 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
11. 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.
12. 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
13. 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.
14. 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.
15. Re-plate surviving colonies in the same manner as step 14, and incubate at 30C for 7 days, until colonies reach ~2cm in diameter
16. 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
17. 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.
18. 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
19. 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