Transfections for gene delivery and genome editing in S. rosetta (VERSION 4)

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

Two days prior to transfection, inoculate 80mL 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. Also add 400µLof 10 of E. pacifica to the flask to provide ample food for growth.

Grow the culture for 40h 0m 0s in a 300 cm² flask at 22°C.

Prepare expression plasmids for transfection

Use standard DNA plasmid preparation protocols to purify plasmids from E. coli that lack adenine and cytidine methyl transferases (dam-/dcm-).

If needed, concentrate DNA plasmids to a final concentration of 5µg/µL using a standard ethanol precipitation.

Prepare a mixture of carrier molecules to add to the purified plasmid.

For one transfection, add 2µL 20µg/µL that has been resuspended in 10millimolar (mM) 8.0

For one transfection, add 1µL 250millimolar (mM) 7.5.

For one transfection, add 1µL 100.

Thoroughly mix the viscous solution by pipetting up and down.

Combine expression plasmids and the carrier mixture for each transfection.

For one transfection, place 4µL of the carrier mixture ( ) in the bottom of a 1.5 ml conical tube.

Add 1µLof 5µg/µL ( ) to the carrier mixture and slowly pipette up and down to thoroughly mix the solution. This solution is called the "Plasmid Delivery Mix."

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 (30millimolar (mM)``7.5 ; 100millimolar (mM)) 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 gRNA concentration of 100micromolar (µM) (gRNA 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 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 while the Sp Cas9/gRNA complex assembles.

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).

µL 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 of100micromolar (µM) in dilution buffer (50millimolar (mM) 7.5 ,200millimolar (mM) , 20% volume and 10millimolar (mM)) from a stock solution of 1millimolar (mM) (the recommended Papain is already at this concentration), and incubate at Room temperature just before priming cells for transfection.

Note

The dilution buffer should be sterile filtered through a 0.22 µm filter. This buffer may also be prepared ahead of time and stored in a -80°C freezer until just before its use.

Prepare the remaining components of the priming buffer (40millimolar (mM) 7.5 ,34millimolar (mM) , 15Mass / % volume and 50millimolar (mM)). DO NOT combine the papain and priming buffer until 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.

Homogenize 80mL culture of S. rosetta feeding on E. pacifica () by vigorously shaking the flask and then splitting the culture into 40mLaliquots in 50 ml conical tubes. Vigorously shake the tubes for 30 sec to further break up bacterial clumps and loosely associated S. rosetta cells

Centrifuge the cells at 2400x g,4°C in a swinging bucket rotor.

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.

Resuspended the two cell pellets in a total volume of 25mL, combine into one conical tube, and homogenize the cells by vigorously shaking the tube for 30 sec.

For a second time, centrifuge the resuspended cells at 2400x g,4°C in a swinging bucket rotor.

Remove the supernatant as before ( ).

Resuspend the cell pellet in 100µL . This resuspension is called the "washed cells."

Prepare aliquots of . 100µLaliquots of 5*10^7.

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

Fix the diluted cells with 2µL 37.5Mass / % volume and homogenize by vortexing.

Pipette 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" with Cell Wash Buffer to final concentration of 5*10^7 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 2m 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 (40millimolar (mM) 7.5 ,34millimolar (mM) , 15Mass / % volume , 50millimolar (mM), and 1.5micromolar (µM))

Resuspend each cell pellet in 100µLof priming buffer.

Incubate cells for 0h 45m 0s at 22Room temperature .

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

Centrifuge cells at 1200x g,22°C .

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 cargo via nucleofection.

For one transfection, add 16µLof ice-cold SF Buffer to the Plasmid Delivery Mix ( ) or the Sp Cas9 RNP ( ).

For genome editing, add 2µL of the repair oligonucleotide template to the Sp Cas9 RNP and SF Buffer ( ).

Add 2µLof "primed cells" (from ) to the tube with delivery cargo and SF Buffer. This solution, which is called the "nucleofection mix."

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

Pulse the nucleofection plate with either the CU 154 (harsher) or the CT 151 (milder) 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 (10millimolar (mM) 7.5 ,900millimolar (mM) , and 8Mass / % volume ) to each 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 2mL in one well of a 6- well cell culture plate.

Incubate at 27°C for 0h 30m 0s

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

Add 10µLof 10 of E. pacifica to the wells in the 6-well plate. For a 12-well plate, only add 5 µl.

Incubate the cell culture plate at 27°C for downstream experiments.