TITLE: Genome editing in the choanoflagellate Salpingoeca rosetta
AUTHORS: David Booth

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details the preparation and execution of CRISPR/Cas9 genome editing in </span><span style = "font-style:italic;">S. rosetta</span><span>.  The protocol builds on a </span><a href="https://www.protocols.io/view/transfection-of-salpingoeca-rosetta-h8bb9sn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">method</span></a><a href="https://www.protocols.io/view/transfection-of-salpingoeca-rosetta-h8bb9sn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> to transfect macromolecules into </span><span style = ":;font-style:italic;">S. rosetta</span></a><span> for delivering a purified Cas9 ribonucleoprotein from </span><span style = "font-style:italic;">Streptomyces pyogenes</span><span> (</span><span style = "font-style:italic;">Sp</span><span>Cas9 RNP) into </span><span style = "font-style:italic;">S. rosetta</span><span>. Upon cleaving the </span><span style = "font-style:italic;">S. rosetta</span><span> genome at locations specified by the guide RNA (gRNA) of the </span><span style = "font-style:italic;">Sp</span><span>Cas9 RNP, </span><span style = "font-style:italic;">S. rosetta</span><span> can use DNA oligonucleotides as templates to repair the double-stranded break. Those repair templates can encode foreign sequences and mutations for editing the </span><span style = "font-style:italic;">S. rosetta</span><span> genome, so long as DNA oligonucleotides have >30 bases of sequence that is homologous to both sides of the Cas9 cleavage site. </span></div></div>

[STEPS]
?. [Culture Cells]
Seed a large culture of S. rosetta.
?. [Culture Cells]
Two days prior to transfection, inoculate   of high nutrient media with a culture of S. rosetta feeding on E. pacifica to a final concentration of S. rosetta of .
120 mL
?. [Culture Cells]
Grow the culture for  in a 3-layer flask at .
[with 60% humidity]
?. [Prepare Gene Editing Cargo]
Prepare a guide RNA (gRNA) that binds to SpCas9 and targets DNA by annealing CRISPR RNA (crRNA) with the trans-activating CRISPR RNA (tracrRNA) .
?. [Prepare Gene Editing Cargo]
Resuspend crRNA in duplex buffer (30 mM HEPES-KOH, pH 7.5; 100 mM potassium acetate) to a final concentration of .
?. [Prepare Gene Editing Cargo]
Resuspend tracrRNA in duplex buffer to a final concentration of .
?. [Prepare Gene Editing Cargo]
Mix equal volumes of crRNA ( ) and tracrRNA ( ) to have a final concentration of  gRNA, which is the annealed complex of crRNA and tracrRNA.
?. [Prepare Gene Editing Cargo]
Incubate the gRNA solution  at  in an aluminum block for .
95 °C
?. [Prepare Gene Editing Cargo]
Place the aluminum block was placed at  to slowly cool the gRNA to .
95 Room temperature
25 °C
?. [Prepare Gene Editing Cargo]
Store the gRNA at .
-20 °C
?. [Prepare Gene Editing Cargo]
Prepare DNA oligonucleotides that serve as repair templates after SpCas9 cleavage.
?. [Prepare Gene Editing Cargo]
Dissolve oligonucleotides to a final concentration of  in 10 mM HEPES-KOH, pH 7.5.
?. [Prepare Gene Editing Cargo]
Incubate the dissolved oligonucleotides at  for .
55 °C
?. [Prepare Gene Editing Cargo]
Store oligonucleotides at .
-20 °C
?. [Prepare Gene Editing Cargo]
Before starting nucleofections, ensure that the oligonucleotides are fully dissolved by incubating them at  for , which concurs with the assembly of the SpCas9/gRNA complex.
55 °C
?. [Prepare Gene Editing Cargo]
Assemble SpCas9 with the gRNA to form the SpCas9 RNP.
?. [Prepare Gene Editing Cargo]
For one transfection, place  of  in the bottom of a 0.2 ml PCR tube.
2 µl
[SpCas9]
?. [Prepare Gene Editing Cargo]
Add of   ( ) by slowly pipetting up and down with SpCas9 to gently mix the gRNA together. This solution is called the "SpCas9 ribonucleoprotein (RNP)."
2 µl
[gRNA]
?. [Prepare Gene Editing Cargo]
Incubate the SpCas9 RNP at  for  (roughly the time to complete the preparation of S. rosetta for priming, see below).
55 Room temperature
?. [Prepare transfection Reagents]
Prepare SF Buffer (Lonza) for transfections.
?. [Prepare transfection Reagents]
Add all of buffer B (smaller volume that may also be called supplement 1) to buffer A (larger volume).
?. [Prepare transfection Reagents]
Store on ice until ready for use. The combined buffer can also be stored at 4°C for up to 3 months.
The combined buffer can be stored at 4°C for up to 3 months.
Because the Lonza kits can be so expensive, we recommend aliquoting large volumes of the SF components (900 µl aliquots for buffer A and 200 µl aliquots for buffer B) to prevent SF buffer from spoiling after buffers A and B have been combined.
?. [Prepare transfection Reagents]
Prepare the priming buffer.
?. [Prepare transfection Reagents]
Dilute papain to a final concentration of 100 µM in dilution buffer(50 mM HEPES-KOH pH 7.5, 200 mM sodium chloride, 20% [v/v] glycerol, and 10 mM cysteine) from a stock solution of 1 mM papain (Millipore Sigma, St. Louis, MO; Cat. No. P3125-100MG]), and incubate at room temperature just before priming cells for transfection.
The dilution buffer [50 mM HEPES-KOH pH 7.5, 200 mM sodium chloride, 20% (v/v) glycerol and 10 mM cysteine] should be sterile filtered through a 0.22 µm filter. The dilution buffer may also be prepared ahead of time and stored in a -80°C freezer just before its use.
?. [Prepare transfection Reagents]
Make a solution of the remaining components of the priming buffer (40 mM HEPES-KOH, pH 7.5; 34 mM lithium citrate; 50 mM L-cysteine; 15% [wt/vol] PEG 8000). DO NOT combine the papain and priming buffer unti just before adding the priming buffer to cells.
The priming buffer without papain should be sterile filtered through a 0.22 µm filter. The priming buffer without papain can also be made ahead of time and stored at -80°C until it is used. Be sure that the priming before is warmed to room temperature prior to use.
?. [Wash Cells]
Prepare S. rosetta for transfection by washing away feeder bacteria.
?. [Wash Cells]
Homogenized the  culture of S. rosetta feeding on E. pacifica () by vigorously shaking and then split into aliquots in 50 ml conical tubes.
120 mL
40 mL
?. [Wash Cells]
Vigorously shake the aliquots and centrifuge the cells for  at  and  in a swinging bucket rotor.
Centrifuge: 2000 34
22 °C
?. [Wash Cells]
Use a serological pipette to gently remove from the cell pellet all but 2 ml of the supernatant, which remains cloudy with E. pacifica bacteria. With a fine tip transfer pipette, gently remove the remaining liquid near the pellet.
?. [Wash Cells]
The three cell pellets were resuspended in a total volume of  artificial seawater, combined into one conical tube, and vigorously shaken to homogenize the cells.
50 mL
?. [Wash Cells]
For a second time, the resuspended cells were centrifuged for for  at  and  in a swinging bucket rotor.
Centrifuge: 2000 34
22 °C
?. [Wash Cells]
The supernatant was removed as before (  ).
?. [Wash Cells]
The pellet was resuspended in  of artificial seawater, and the cells were homogenized by vigorous shaking.
50 mL
?. [Wash Cells]
The cells were centrifuged for a third time for  at  and .
Centrifuge: 2200 34
22 °C
?. [Wash Cells]
Remove the supernatant as before (  ).
?. [Wash Cells]
Resuspend the cell pellet in  of artificial seawater. This resuspension is called the "washed cells."
400 µl
?. [Wash Cells]
Prepare aliquots of .
100 µl
?. [Wash Cells]
Dilute  of "washed cells" ( ) into of artificial seawater.
2 µl
196 µl
?. [Wash Cells]
Fix the diluted cells with  of 37.5% formaldehyde and homogenize by vortexing.
2 µl
?. [Wash Cells]
Pipet the fixed cells into a fixed chamber slide and determine the cell concentration.
Remember that concentration of diluted and fixed cells is a 100-fold dilution from the "washed cells." Be sure to factor that dilution into your concentration.
Cells can be counted on a hemacytometer (Neubauer with brightlines) or with an automated cell counter. We recommend a Luna-FL automated cell counter.
?. [Wash Cells]
After determining the cell concentration, dilute the "washed cells" to final concentration of   and split into aliquots.
100 µl
One aliquot provides enough cells for 12 nucleofections.
?. [Prime Cells]
Prime cells for nucleofection by degrading the glycocalyx that surrounds S. rosetta.
?. [Prime Cells]
Spin the  aliquots of washed cells ( ) at  and  for .
100 µl
Centrifuge: 800 34
22 °C
?. [Prime Cells]
Gently remove the supernatant from the cell pellet with a gel-loading pipette tip.
?. [Prime Cells]
Combine the priming buffer components ()  to make a final priming buffer (40 mM HEPES-KOH, pH 7.5; 34 mM lithium citrate; 50 mM l-cysteine; 15% [wt/vol] PEG 8000; and 1 µM papain)
?. [Prime Cells]
Resuspend each cell pellet in of priming buffer.
100 µl
?. [Prime Cells]
Incubate cells for  at  .
22 Room temperature
?. [Prime Cells]
Add  of  to each aliquot of primed cells for quenching proteolysis from the priming buffer.
10 µl
[Bovine Serum Albumin]
?. [Prime Cells]
Centrifuge cells at  and  for  .
Centrifuge: 1250 34
22 °C
?. [Prime Cells]
Gently remove the supernatant from the cell pellet with a gel-loading pipette tip.
?. [Prime Cells]
Resuspended each cell pell in  of SF Buffer (  ). This suspension of cells is called the "primed cells."
25 µl
?. [Prime Cells]
Store the "primed cells" on ice while preparing nucleofection reactions.
?. [Transfect Cells]
Deliver gene editing cargo via nucleofection.
?. [Transfect Cells]
Add of ice-cold SF Buffer ( ) to the SpCas9 RNP ( ), which has a total volume of .
16 µl
4 µl
For reactions that use two different gRNAs, assemble each SpCas9 RNP separately then combine each SpCas9 RNP at this step. After the SpCas9 RNPs have been combined, add   of ice-cold SF Buffer
?. [Transfect Cells]
Add  of the repair oligonucleotide template ( ) to the PCR tube with SpCas9 RNP and SF Buffer (  ).
2 µl
?. [Transfect Cells]
Add of "primed cells" (from  ) to the PCR tube with SpCas9 RNP, SF Buffer, and the repair template (  ). This solution, which is called the "nucleofection mix," should have a total volume of .
2 µl
24 µl
?. [Transfect Cells]
Transfer the entire nucleofection mix into one well of a 96-well nucleofection plate.
At this point, prepare for the recovery step, by transferring the recovery buffer into a convenient vessel and setting the pipette to  .
?. [Transfect Cells]
Pulse the nucleofection plate with the CM156 pulse.
?. [Rest and Recover Cells]
Allow membranes to reseal by resting cells in recovery buffer before growing cells again in media.
?. [Rest and Recover Cells]
Immediately after transfection, add of ice-cold recovery buffer (10 mM HEPES-KOH, pH 7.5; 0.9 M sorbitol; 8% [wt/vol] PEG 8000) to each nucleofection transfection and gently mixed by firmly tapping the side of the plate.
100 µl
?. [Rest and Recover Cells]
Allow cells to rest in recovery buffer for .
?. [Rest and Recover Cells]
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 to  of low nutrient media in one well of a 6 well plate.
2 mL
?. [Rest and Recover Cells]
Incubate at  for
[and 60% humidity]
?. [Rest and Recover Cells]
Add E. pacifica food and grow transfected cells.
?. [Rest and Recover Cells]
Add of  of E. pacifica  to the wells in the 6 well plate.
10 µl
Prepare the E. pacifica solution by resuspending a frozen, 10 mg pellet of E. pacifica in 1 ml of artificial seawater.
?. [Rest and Recover Cells]
Incubate the 6 well plate at  for  before using in downstream experiments.
[and 60% humidity]
?. [(Optional) Select for Cycloheximide Resistance]
Add of of cycloheximide to the culture of transfected cells after allowing the cells to fully recover.
10 µl
2 mL
Cycloheximide is toxic. Handle carefully and properly dispose.
?. [(Optional) Select for Cycloheximide Resistance]
Incubate the cells in cycloheximide for  prior to genotyping and clonal isolation.