TITLE: Transfections for gene delivery and genome editing in S. rosetta (VERSION 4)
AUTHORS: David Booth

[DESCRIPTION]
This protocol details the preparation and execution of CRISPR/Cas9 genome editing in S. rosetta.  The protocol builds on a method to transfect macromolecules into S. rosetta for delivering a purified Cas9 ribonucleoprotein from Streptomyces pyogenes (SpCas9 RNP) into S. rosetta. Upon cleaving the S. rosetta genome at locations specified by the guide RNA (gRNA) of the SpCas9 RNP, S. rosetta can use DNA oligonucleotides as templates to repair the double-stranded break. Those repair templates can encode foreign sequences and mutations for editing the S. rosetta genome, so long as DNA oligonucleotides have >30 bases of sequence that is homologous to both sides of the Cas9 cleavage site. 

[BEFORE_START]
Please consult the attached file of media recipes for artificial seawater, high nutrient media, and low nutrient media.  


[GUIDELINES]
Perform cell culturing and transfection procedure inside of a biosafety cabinet to maintain sterility. 

[STEPS]
SECTION: Culture Cells
1. Seed a large culture of S. rosetta.

SECTION: Culture Cells
1.1. Two days prior to transfection, inoculate  80 mL of media (15% Red Algae + 2% Peptone-Yeast-Glycerol) with a culture of S. rosetta feeding on E. pacifica (ATCC PRA-390) to a final concentration of S. rosetta of 1.5*10^4 cells/ml.  Also add 400 µLof 10 mg/ml of E. pacifica  to the flask to provide ample food for growth.

 


SECTION: Culture Cells
1.2. Grow the culture for 2400 min in a 300 cm2 flask at 22 °C.

SECTION: Prepare cargo for transgene expression from plasmids
2. Prepare expression plasmids for transfection
 

SECTION: Prepare cargo for transgene expression from plasmids
2.1. Use standard DNA plasmid preparation protocols to purify plasmids from E. coli that lack adenine and cytidine methyl transferases (dam-/dcm-). 
SECTION: Prepare cargo for transgene expression from plasmids
2.2. 
If needed, concentrate DNA plasmids to a final concentration of 5 µg/µL using a standard ethanol precipitation.  
 

 


SECTION: Prepare cargo for transgene expression from plasmids
3. Prepare a mixture of carrier molecules to add to the purified plasmid.

 

SECTION: Prepare cargo for transgene expression from plasmids
3.1. For one transfection, add 2 µL 20 µg/µL that has been resuspended in 10 millimolar (mM) pH 8.0 

SECTION: Prepare cargo for transgene expression from plasmids
3.2. For one transfection, add 1 µL 250 millimolar (mM)  pH 7.5.

SECTION: Prepare cargo for transgene expression from plasmids
3.3. For one transfection, add 1 µL 100 mg/ml.
SECTION: Prepare cargo for transgene expression from plasmids
3.4. Thoroughly mix the viscous solution by pipetting up and down. 
SECTION: Prepare cargo for transgene expression from plasmids
4. Combine expression plasmids and the carrier mixture for each transfection.  

 

SECTION: Prepare cargo for transgene expression from plasmids
4.1. For one transfection, place 4 µL of the carrier mixture (  ) in the bottom of a 1.5 ml conical tube.
SECTION: Prepare cargo for transgene expression from plasmids
4.2. Add 1 µLof 5 µg/µL  (  ) to the carrier mixture and slowly pipette up and down to thoroughly mix the solution. This solution is called the "Plasmid Delivery Mix."

 


SECTION: Prepare cargo for genome editing with Cas9 RNP
5. Prepare a guide RNA (gRNA) that binds to SpCas9 and targets DNA by annealing CRISPR RNA (crRNA) with the trans-activating CRISPR RNA (tracrRNA) . 
SECTION: Prepare cargo for genome editing with Cas9 RNP
5.1. Resuspend crRNA in duplex buffer (30 millimolar (mM)pH 7.5 ; 100 millimolar (mM)) to a final concentration of 200 micromolar (µM).

SECTION: Prepare cargo for genome editing with Cas9 RNP
5.2. Resuspend tracrRNA in duplex buffer to a final concentration of 200 micromolar (µM).

SECTION: Prepare cargo for genome editing with Cas9 RNP
5.3. Mix equal volumes of crRNA (  ) and tracrRNA (  ) to have a final gRNA concentration of 100 micromolar (µM) (gRNA is the annealed complex of crRNA and tracrRNA). 
SECTION: Prepare cargo for genome editing with Cas9 RNP
5.4. Incubate the gRNA solution  at 95 °C in an aluminum block for 5 min.
SECTION: Prepare cargo for genome editing with Cas9 RNP
5.5. Place the aluminum block at 95 Room temperature to slowly cool the gRNA to 25 °C.
SECTION: Prepare cargo for genome editing with Cas9 RNP
5.6. Store the gRNA at -20 °C.

SECTION: Prepare cargo for genome editing with Cas9 RNP
6. Prepare DNA oligonucleotides that serve as repair templates after SpCas9 cleavage. 
SECTION: Prepare cargo for genome editing with Cas9 RNP
6.1. Dissolve oligonucleotides to a final concentration of 250 micromolar (µM) in 10 mM HEPES-KOH, pH 7.5. 
SECTION: Prepare cargo for genome editing with Cas9 RNP
6.2. Incubate the dissolved oligonucleotides at 55 °C for 60 min.
SECTION: Prepare cargo for genome editing with Cas9 RNP
6.3. Store oligonucleotides at -20 °C.

SECTION: Prepare cargo for genome editing with Cas9 RNP
6.4. Before starting nucleofections, ensure that the oligonucleotides are fully dissolved by incubating them at 55 °C for 60 min, which concurs while the SpCas9/gRNA complex assembles.

SECTION: Prepare cargo for genome editing with Cas9 RNP
7. Assemble SpCas9 with the gRNA to form the SpCas9 RNP. 
SECTION: Prepare cargo for genome editing with Cas9 RNP
7.1. For one transfection, place 2 µL of 20 micromolar (µM) in the bottom of a 0.2 ml PCR tube.
SECTION: Prepare cargo for genome editing with Cas9 RNP
7.2. Add 2 µLof 100 micromolar (µM)  (  ) by slowly pipetting up and down with SpCas9 to gently mix the gRNA together. This solution is called the "SpCas9 ribonucleoprotein (RNP)." 
SECTION: Prepare cargo for genome editing with Cas9 RNP
7.3. Incubate the SpCas9 RNP at 55 Room temperature for 60 min (roughly the time to complete the preparation of S. rosetta for priming, see below). 

SECTION: Prepare transfection Reagents
8. Prepare SF Buffer (Lonza) for transfections.
SECTION: Prepare transfection Reagents
8.1. Add all of buffer B (smaller volume that may also be called supplement 1) to buffer A (larger volume).
SECTION: Prepare transfection Reagents
8.2.   Store on ice until ready for use. The combined buffer can also be stored at 4°C for up to 3 months. 

 

 

SECTION: Prepare transfection Reagents
9. Prepare the priming buffer.
SECTION: Prepare transfection Reagents
9.1. Dilute papain to a final concentration of100 micromolar (µM) in dilution buffer (50 millimolar (mM) pH 7.5 ,200 millimolar (mM) , 20 % volume  and 10 millimolar (mM)) from a stock solution of 1 millimolar (mM) (the recommended Papain is already at this concentration),  and incubate at Room temperature just before priming cells for transfection. 
 

 




SECTION: Prepare transfection Reagents
9.2. Prepare the remaining components of the priming buffer (40 millimolar (mM) pH 7.5 ,34 millimolar (mM) , 15 Mass / % volume  and 50 millimolar (mM)). DO NOT combine the papain and priming buffer until just before adding the priming buffer to cells. 

 

SECTION: Wash Cells
10. Prepare S. rosetta for transfection by washing away feeder bacteria. 
SECTION: Wash Cells
10.1. Homogenize  80 mL culture of S. rosetta feeding on E. pacifica ( ) by vigorously shaking the flask and then splitting the culture into 40 mLaliquots in 50 ml conical tubes. Vigorously shake the tubes for 30 sec to further break up bacterial clumps and loosely associated S. rosetta cells
SECTION: Wash Cells
10.2. Centrifuge the cells at 2400 x g, 3 min, 4 °C in a swinging bucket rotor.
SECTION: Wash Cells
10.3. Use a serological pipette to gently remove all but 2 ml of the supernatant from the cell pellet. With a fine tip transfer pipette, gently remove the remaining liquid near the pellet. Note that the supernatant may probably be cloudy with E. pacifica bacteria. 
SECTION: Wash Cells
10.4. Resuspended the two cell pellets in a total volume of 25 mL, combine into one conical tube, and homogenize the cells by vigorously shaking the tube for 30 sec.

 

SECTION: Wash Cells
10.5. For a second time, centrifuge the resuspended cells at 2400 x g, 3 min, 4 °C in a swinging bucket rotor.

SECTION: Wash Cells
10.6. Remove the supernatant as before (   ).
SECTION: Wash Cells
10.7. Resuspend the cell pellet in 100 µL . This resuspension is called the "washed cells." 
SECTION: Wash Cells
11. Prepare 100 µLaliquots of 5*10^7 cells/ml.
SECTION: Wash Cells
11.1. Dilute 2 µL of "washed cells" (  ) into 196 µL.

SECTION: Wash Cells
11.2. Fix the diluted cells with 2 µL 37.5 Mass / % volume and homogenize by vortexing.
SECTION: Wash Cells
11.3. Pipette the fixed cells into a fixed chamber slide and determine the cell concentration. 
 


 

 


SECTION: Wash Cells
11.4. After determining the cell concentration, dilute the "washed cells"  with Cell Wash Buffer to final concentration of 5*10^7 cells/ml  and split into 100 µLaliquots.

 

SECTION: Prime Cells
12. Prime cells for nucleofection by degrading the glycocalyx that surrounds S. rosetta.  
SECTION: Prime Cells
12.1. Spin the 100 µL aliquots of washed cells (  ) at 800 x g and 22 °C for 2 min.
SECTION: Prime Cells
12.2. Gently remove the supernatant from the cell pellet with a gel-loading pipette tip. 
SECTION: Prime Cells
12.3. Combine the priming buffer components ( )  to make a final priming buffer (40 millimolar (mM) pH 7.5 ,34 millimolar (mM) , 15 Mass / % volume ,  50 millimolar (mM), and  1.5 micromolar (µM))
SECTION: Prime Cells
12.4. Resuspend each cell pellet in 100 µLof priming buffer.
SECTION: Prime Cells
12.5. Incubate cells for 45 min at 22 Room temperature .
SECTION: Prime Cells
12.6. Add 10 µL of 50 mg/ml to each aliquot of primed cells for quenching proteolysis from the priming buffer.
SECTION: Prime Cells
12.7. Centrifuge cells at 1200 x g, 4 min, 22 °C . 
SECTION: Prime Cells
12.8. Gently remove the supernatant from the cell pellet with a gel-loading pipette tip.
SECTION: Prime Cells
12.9. Resuspended each cell pell in 25 µL of SF Buffer (   ). This suspension of cells is called the "primed cells."


SECTION: Prime Cells
12.10. Store the "primed cells" on ice while preparing nucleofection reactions. 
SECTION: Transfect Cells
13. Deliver cargo via nucleofection.
SECTION: Transfect Cells
13.1. For one transfection, add 16 µLof ice-cold SF Buffer to the Plasmid Delivery Mix (  ) or the SpCas9 RNP (  ).

 

SECTION: Transfect Cells
13.2. For genome editing, add 2 µL of the repair oligonucleotide template to the SpCas9 RNP and SF Buffer (   ).
SECTION: Transfect Cells
13.3. Add 2 µLof "primed cells" (from   ) to the tube with delivery cargo and SF Buffer. This solution, which is called the "nucleofection mix."
SECTION: Transfect Cells
13.4. Transfer the entire nucleofection mix into one well of a 96-well nucleofection plate. 

 

SECTION: Transfect Cells
13.5. Pulse the nucleofection plate with either the CU 154 (harsher) or the CT 151 (milder) pulse. 

 


 

SECTION: Rest and Recover Cells
14. Allow membranes to reseal by resting cells in recovery buffer before growing cells again in media.  
SECTION: Rest and Recover Cells
14.1. Immediately after transfection, add 100 µLof ice-cold recovery buffer (10 millimolar (mM) pH 7.5 ,900 millimolar (mM) , and  8 Mass / % volume ) to each transfection and gently mixed by firmly tapping the side of the plate.
 

SECTION: Rest and Recover Cells
14.2. Allow cells to rest in recovery buffer for 5 min. 
SECTION: Rest and Recover Cells
14.3. Gently mix the well in the nucleofection plate by pipetting up and down before transferring the entire volume in nucleofection well (the nucleofection mix plus the recovery buffer) into 2 mL in one well of a 6- well cell culture plate. 
 



SECTION: Rest and Recover Cells
14.4. Incubate at 27 °C for 30 min 
SECTION: Rest and Recover Cells
15. Add E. pacifica food and grow transfected cells. 
SECTION: Rest and Recover Cells
15.1. Add 10 µLof 10 mg/ml of E. pacifica  to the wells in the 6-well plate. For a 12-well plate, only add 5 µl. 

 

SECTION: Rest and Recover Cells
15.2. Incubate the cell culture plate at 27 °C for downstream experiments.