Sep 11, 2021
Version 2
Genome editing in the choanoflagellate Salpingoeca rosetta V.2
- 1University of California, San Francisco
- King Lab
- Protist Research to Optimize Tools in Genetics (PROT-G)
Protocol Citation: David Booth 2021. Genome editing in the choanoflagellate Salpingoeca rosetta. protocols.io https://dx.doi.org/10.17504/protocols.io.bx58pq9wVersion created by David Booth
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 11, 2021
Last Modified: September 11, 2021
Protocol Integer ID: 53152
Abstract
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.
Guidelines
Perform cell culturing and transfection procedure inside of a biosafety cabinet to maintain sterility.
Materials
STEP MATERIALS
DNA OligonucleotideIntegrated DNA TechnologiesCatalog #Custom Order
tracrRNAIntegrated DNA TechnologiesCatalog #1072534
Falcon 525cm² Rectangular Straight Neck Cell Culture Multi-Flask, 3-layer with Vented CapCorningCatalog #353143
EnGen Cas9 NLS, S. pyogenes - 400 pmolNew England BiolabsCatalog #M0646T
SF Cell Line 96-well Nucleofector KitLonzaCatalog #V4SC-2096
Salpingoeca rosetta cultured with Echinicola pacifica (SrEpac)ATCCCatalog #PRA-390
crRNAIntegrated DNA TechnologiesCatalog #Custom Order
Duplex BufferIntegrated DNA TechnologiesCatalog #11-01-03-01
Papain from papaya latexMillipore SigmaCatalog #P3125-100MG
Protocol materials
tracrRNAIntegrated DNA Technologies, Inc. (IDT)Catalog #1072534
EnGen Cas9 NLS, S. pyogenes - 400 pmolNew England BiolabsCatalog #M0646T
crRNAIntegrated DNA Technologies, Inc. (IDT)Catalog #Custom Order
Duplex BufferIntegrated DNA Technologies, Inc. (IDT)Catalog #11-01-03-01
Papain from papaya latexMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3125-100MG
DNA OligonucleotideIntegrated DNA Technologies, Inc. (IDT)Catalog #Custom Order
Falcon 525cm² Rectangular Straight Neck Cell Culture Multi-Flask, 3-layer with Vented CapCorningCatalog #353143
SF Cell Line 96-well Nucleofector KitLonzaCatalog #V4SC-2096
Salpingoeca rosetta cultured with Echinicola pacifica (SrEpac)ATCCCatalog #PRA-390
Salpingoeca rosetta cultured with Echinicola pacifica (SrEpac)ATCCCatalog #PRA-390
Falcon 525cm² Rectangular Straight Neck Cell Culture Multi-Flask, 3-layer with Vented CapCorningCatalog #353143
crRNAIntegrated DNA Technologies, Inc. (IDT)Catalog #Custom Order
Duplex BufferIntegrated DNA Technologies, Inc. (IDT)Catalog #11-01-03-01
tracrRNAIntegrated DNA Technologies, Inc. (IDT)Catalog #1072534
DNA OligonucleotideIntegrated DNA Technologies, Inc. (IDT)Catalog #Custom Order
EnGen Cas9 NLS, S. pyogenes - 400 pmolNew England BiolabsCatalog #M0646T
SF Cell Line 96-well Nucleofector KitLonzaCatalog #V4SC-2096
Papain from papaya latexMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3125-100MG
Before start
Please consult the attached file of media recipes for artificial seawater, high nutrient media, and low nutrient media.
MediaRecipes.pdf
MediaRecipes.pdf Culture Cells
Culture Cells
Seed a large culture of S. rosetta.
Two days prior to transfection, inoculate 120 mL of high nutrient media with a culture of S. rosetta feeding on E. pacifica to a final concentration of S. rosetta of 8000 cells/ml .
Salpingoeca rosetta cultured with Echinicola pacifica (SrEpac)Sigma AldrichCatalog #PRA-390
Grow the culture for 48:00:00 in a 3-layer flask at 22 °C with 60% humidity .
Falcon 525cm² Rectangular Straight Neck Cell Culture Multi-Flask, 3-layer with Vented CapSigma AldrichCatalog #353143
Prepare Gene Editing Cargo
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) .
Resuspend crRNA in duplex buffer (30 mM HEPES-KOH, pH 7.5; 100 mM potassium acetate) to a final concentration of 200 micromolar (µM) .
crRNASigma AldrichCatalog #Custom Order
Duplex BufferSigma AldrichCatalog #11-01-03-01
Resuspend tracrRNA in duplex buffer to a final concentration of 200 micromolar (µM) .
tracrRNASigma AldrichCatalog #1072534
Mix equal volumes of crRNA (go to step #2.1 ) and tracrRNA (go to step #2.2 ) to have a final concentration of 100 micromolar (µM) gRNA, which is the annealed complex of crRNA and tracrRNA.
Incubate the gRNA solution at 95 °C in an aluminum block for 00:05:00 .
Place the aluminum block was placed at Room 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.
Dissolve oligonucleotides to a final concentration of 250 micromolar (µM) in 10 mM HEPES-KOH, pH 7.5.
DNA OligonucleotideSigma AldrichCatalog #Custom Order
Incubate the dissolved oligonucleotides at 55 °C for 01:00:00 .
Store oligonucleotides at -20 °C .
Before starting nucleofections, ensure that the oligonucleotides are fully dissolved by incubating them at 55 °C for 01:00:00 , which concurs with the assembly of the SpCas9/gRNA complex.
Assemble SpCas9 with the gRNA to form the SpCas9 RNP.
For one transfection, place 2 µL of 20 micromolar (µM) SpCas9 in the bottom of a 0.2 ml PCR tube.
EnGen Cas9 NLS, S. pyogenes - 400 pmolSigma AldrichCatalog #M0646T
Add 2 µL of 100 micromolar (µM) gRNA (go to step #2 ) by slowly pipetting up and down with SpCas9 to gently mix the gRNA together. This solution is called the "SpCas9 ribonucleoprotein (RNP)."
Incubate the SpCas9 RNP at Room temperature for 01:00:00 (roughly the time to complete the preparation of S. rosetta for priming, see below).
Prepare transfection Reagents
Prepare transfection Reagents
Prepare SF Buffer (Lonza) for transfections.
SF Cell Line 96-well Nucleofector KitSigma AldrichCatalog #V4SC-2096
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.
Note
The combined buffer can be stored at 4°C for up to 3 months.
Note
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 the 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.
Papain from papaya latexSigma AldrichCatalog #P3125-100MG
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.
Note
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
Wash Cells
Prepare S. rosetta for transfection by washing away feeder bacteria.
Homogenized the 120 mL culture of S. rosetta feeding on E. pacifica (go to step #1 ) by vigorously shaking and then split into 40 mL aliquots in 50 ml conical tubes.
Vigorously shake the aliquots and centrifuge the cells for 00:05:00 at 2000 x 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 50 mL 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 00:05:00 at 2000 x g and 22 °C in a swinging bucket rotor.
The supernatant was removed as before ( go to step #7.3 ).
The pellet was resuspended in 50 mL of artificial seawater, and the cells were homogenized by vigorous shaking.
The cells were centrifuged for a third time for 00:05:00 at 2200 x g and 22 °C .
Remove the supernatant as before ( go to step #7.3 ).
Resuspend the cell pellet in 400 µL of artificial seawater. This resuspension is called the "washed cells."
Prepare 100 µL aliquots of 50000000 cells/ml .
Dilute 2 µL of "washed cells" (go to step #7.10 ) into 196 µL of artificial seawater.
Fix the diluted cells with 2 µL of 37.5% formaldehyde and homogenize by vortexing.
Pipet the fixed cells into a fixed chamber slide and determine the cell concentration.
Note
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.
Note
Cells can be counted on a hemacytometer (Neubauer with brightlines) or with an automated cell counter. We recommend a Luna-FL automated cell counter.
Equipment
LUNA-FL
NAME
Dual Fluorescence Cell Counter
TYPE
Logos Biosystems
BRAND
L20001
SKU
LINK
After determining the cell concentration, dilute the "washed cells" to final concentration of 50000000 cells/ml and split into 100 µL aliquots.
Note
One aliquot provides enough cells for 12 nucleofections.
Prime Cells
Prime Cells
Prime cells for nucleofection by degrading the glycocalyx that surrounds S. rosetta.
Spin the 100 µL aliquots of washed cells (go to step #8.4 ) at 800 x g and 22 °C for 00:05:00 .
Gently remove the supernatant from the cell pellet with a gel-loading pipette tip.
Combine the priming buffer components (go to step #6 ) 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 100 µL of priming buffer.
Incubate cells for 00:35:00 at Room temperature .
Add 10 µL of 50 mg/ml Bovine Serum Albumin to each aliquot of primed cells for quenching proteolysis from the priming buffer.
Centrifuge cells at 1250 x g and 22 °C for 00:05:00 .
Gently remove the supernatant from the cell pellet with a gel-loading pipette tip.
Resuspended each cell pell in 25 µL of SF Buffer ( go to step #5 ). This suspension of cells is called the "primed cells."
Store the "primed cells" on ice while preparing nucleofection reactions.
Transfect Cells
Transfect Cells
Deliver gene editing cargo via nucleofection.
Add 16 µL of ice-cold SF Buffer (go to step #5 ) to the SpCas9 RNP (go to step #4.3 ), which has a total volume of 4 µL .
Note
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 16 µL of ice-cold SF Buffer
Add 2 µL of the repair oligonucleotide template (go to step #3.4 ) to the PCR tube with SpCas9 RNP and SF Buffer ( go to step #10.1 ).
Add 2 µL of "primed cells" (from go to step #9.10 ) to the PCR tube with SpCas9 RNP, SF Buffer, and the repair template ( go to step #10.2 ). This solution, which is called the "nucleofection mix," should have a total volume of 24 µL .
Transfer the entire nucleofection mix into one well of a 96-well nucleofection plate.
Note
At this point, prepare for the recovery step, by transferring the recovery buffer into a convenient vessel and setting the pipette to 100 µL .
Pulse the nucleofection plate with the CM156 pulse.
Equipment
4D-Nucleofector Core Unit
NAME
Control system for performing nucleofection
TYPE
Lonza
BRAND
AAF-1002B
SKU
LINK
Equipment
96-well Shuttle Device
NAME
Add-on for Nucelofector 4d device to perform plate-based nucleofections
TYPE
Lonza
BRAND
AAM-1001S
SKU
https://bioscience.lonza.com/lonza_bs/US/en/Transfection/p/000000000000191639/96-well-Shuttle-Device
LINK
Rest and Recover Cells
Rest and Recover Cells
Allow membranes to reseal by resting cells in recovery buffer before growing cells again in media.
Immediately after transfection, add 100 µL 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.
Allow cells to rest in recovery buffer for 00:05:00 .
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 2 mL of low nutrient media in one well of a 6 well plate.
Incubate at 22 °C and 60% humidity for 00:30:00
Add E. pacifica food and grow transfected cells.
Add 10 µL of 10 mg/ml of E. pacifica to the wells in the 6 well plate.
Note
Prepare the E. pacifica solution by resuspending a frozen, 10 mg pellet of E. pacifica in 1 ml of artificial seawater.
Incubate the 6 well plate at 22 °C and 60% humidity for 24:00:00 before using in downstream experiments.
(Optional) Select for Cycloheximide Resistance
(Optional) Select for Cycloheximide Resistance
Add 10 µL of 1 µg/ml of cycloheximide to the 2 mL culture of transfected cells after allowing the cells to fully recover.
Safety information
Cycloheximide is toxic. Handle carefully and properly dispose.
Incubate the cells in cycloheximide for 96:00:00 prior to genotyping and clonal isolation.