Genome editing in the choanoflagellate Salpingoeca rosetta

Seed a large culture of S. rosetta. S. rosetta .

Two days prior to transfection, inoculate 120mL of high nutrient media with a culture of S. rosetta feeding on E. pacifica to a final concentration of S. rosetta of 8000cells/ml.

Grow the culture for 48h 0m 0s in a 3-layer flask at 22°C.

Prepare a guide RNA (gRNA) that binds to SpCas9 and targets DNA by annealing CRISPR RNA (crRNA) with the trans-activating CRISPR RNA (tracrRNA) . Sp Cas9 and targets DNA by annealing CRISPR RNA (crRNA) with the trans-activating CRISPR RNA (tracrRNA) .

Resuspend crRNA in duplex buffer (30 mM HEPES-KOH, pH 7.5; 100 mM potassium acetate) to a final concentration of 200micromolar (µM).

Resuspend tracrRNA in duplex buffer to a final concentration of 200micromolar (µM).

Mix equal volumes of crRNA ( ) and tracrRNA ( ) to have a final concentration of 100micromolar (µM) gRNA, which is the annealed complex of crRNA and tracrRNA.

Incubate the gRNA solution at 95°C in an aluminum block for 0h 5m 0s.

Place the aluminum block was placed at 95Room temperature to slowly cool the gRNA to 25°C.

Store the gRNA at -20°C.

Prepare DNA oligonucleotides that serve as repair templates after SpCas9 cleavage. Sp Cas9 cleavage.

Dissolve oligonucleotides to a final concentration of 250micromolar (µM) in 10 mM HEPES-KOH, pH 7.5.

Incubate the dissolved oligonucleotides at 55°C for 1h 0m 0s.

Store oligonucleotides at -20°C.

Before starting nucleofections, ensure that the oligonucleotides are fully dissolved by incubating them at 55°C for 1h 0m 0s, which concurs with the assembly of the Sp Cas9/gRNA complex.

Assemble SpCas9 with the gRNA to form the SpCas9 RNP. Sp Cas9 with the gRNA to form the Sp Cas9 RNP.

For one transfection, place 2µL of 20micromolar (µM) in the bottom of a 0.2 ml PCR tube.

Add 2µLof 100micromolar (µM) ( ) by slowly pipetting up and down with Sp Cas9 to gently mix the gRNA together. This solution is called the " Sp Cas9 ribonucleoprotein (RNP)."

Incubate the Sp Cas9 RNP at 55Room temperature for 1h 0m 0s (roughly the time to complete the preparation of S. rosetta for priming, see below).

Prepare SF Buffer (Lonza) for transfections. SF Buffer (Lonza) for transfections.

Add all of buffer B (smaller volume that may also be called supplement 1) to buffer A (larger volume).

Store on ice until ready for use. The combined buffer can also be stored at 4°C for up to 3 months.

Prepare the priming buffer. priming buffer.

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.

Note

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.

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.

Prepare S. rosetta for transfection by washing away feeder bacteria. S. rosetta for transfection by washing away feeder bacteria.

Homogenized the 120mL culture of S. rosetta feeding on E. pacifica () by vigorously shaking and then split into 40mLaliquots in 50 ml conical tubes.

Resuspend the cell pellet in 400µL of artificial seawater. This resuspension is called the "washed cells."

Vigorously shake the aliquots and centrifuge the cells for 0h 5m 0s at 2000x g and 22°C in a swinging bucket rotor.

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.

The three cell pellets were resuspended in a total volume of 50mL artificial seawater, combined into one conical tube, and vigorously shaken to homogenize the cells.

For a second time, the resuspended cells were centrifuged for for 0h 5m 0s at 2000x g and 22°C in a swinging bucket rotor.

The supernatant was removed as before ( ).

The pellet was resuspended in 50mL of artificial seawater, and the cells were homogenized by vigorous shaking.

The cells were centrifuged for a third time for 0h 5m 0s at 2200x g and 22°C.

Remove the supernatant as before ( ).

Prepare aliquots of . 100µLaliquots of 50000000cells/ml.

Dilute 2µL of "washed cells" ( ) into 196µLof artificial seawater.

Fix the diluted cells with 2µL of 37.5% formaldehyde and homogenize by vortexing.

Pipet the fixed cells into a fixed chamber slide and determine the cell concentration.

Equipment

Value	Label
LUNA-FL	NAME
Dual Fluorescence Cell Counter	TYPE
Logos Biosystems	BRAND
L20001	SKU

After determining the cell concentration, dilute the "washed cells" to final concentration of 50000000cells/ml and split into 100µLaliquots.

Prime cells for nucleofection by degrading the glycocalyx that surrounds S. rosetta. S. rosetta .

Spin the aliquots of washed cells 100µL aliquots of washed cells ( ) at 800x g and 22°C for 0h 5m 0s.

Store the "primed cells" on ice while preparing nucleofection reactions.

Gently remove the supernatant from the cell pellet with a gel-loading pipette tip.

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)

Resuspend each cell pellet in 100µLof priming buffer.

Incubate cells for 0h 35m 0s at 22Room temperature .

Add 10µL of 50mg/ml to each aliquot of primed cells for quenching proteolysis from the priming buffer.

Centrifuge cells at 1250x g and 22°C for 0h 5m 0s .

Gently remove the supernatant from the cell pellet with a gel-loading pipette tip.

Resuspended each cell pell in 25µL of SF Buffer ( ). This suspension of cells is called the "primed cells."

Deliver gene editing cargo via nucleofection.

Add 16µLof ice-cold SF Buffer ( ) to the Sp Cas9 RNP ( ), which has a total volume of 4µL.

Add 2µL of the repair oligonucleotide template ( ) to the PCR tube with Sp Cas9 RNP and SF Buffer ( ).

Add 2µLof "primed cells" (from ) to the PCR tube with Sp Cas9 RNP, SF Buffer, and the repair template ( ). This solution, which is called the "nucleofection mix," should have a total volume of 24µL.

Transfer the entire nucleofection mix into one well of a 96-well nucleofection plate.

Pulse the nucleofection plate with the CM156 pulse.

Equipment

Value	Label
4D-Nucleofector Core Unit	NAME
Control system for performing nucleofection	TYPE
Lonza	BRAND
AAF-1002B	SKU

Equipment

Value	Label
96-well Shuttle Device	NAME
Add-on for Nucelofector 4d device to perform plate-based nucleofections	TYPE
Lonza	BRAND
AAM-1001S	SKU

Allow membranes to reseal by resting cells in recovery buffer before growing cells again in media.

Immediately after transfection, add 100µLof 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.

Allow cells to rest in recovery buffer for 0h 5m 0s.

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 2mL of low nutrient media in one well of a 6 well plate.

Incubate at 22°C for 0h 30m 0s

Add E. pacifica food and grow transfected cells. E. pacifica food and grow transfected cells.

Add 10µLof 10mg/ml of E. pacifica to the wells in the 6 well plate.

Incubate the 6 well plate at 22°C for 24h 0m 0s before using in downstream experiments.

Add 10µLof 1µg/mlof cycloheximide to the 2mLculture of transfected cells after allowing the cells to fully recover.

Incubate the cells in cycloheximide for 96h 0m 0s prior to genotyping and clonal isolation.