Materials and Reagents

Pipette tips, 100-1,250 μl (VWR, catalog number: 89079-470 )
Pipette tips, 1-200 μl (VWR, catalog number: 89079-478 )
Pipette tips, 0.1-10 μl (VWR, catalog number: 89079-464 )
1.5 ml Eppendorf tubes (Corning, Axygen^®, catalog number: MCT-150-C )
0.2 ml PCR tubes (VWR, catalog number: 20170-012 )
Parafilm (Bemis, catalog number: PM996 )
50 ml centrifuge tubes (Greiner Bio One, catalog number: 227261 )
Nalgene^TM filters (Thermo Fisher Scientific, catalog number: 595-4520 )
Phaeodactylum tricornutum cells [Culture Collection of Algae and Protozoa (CCAP, UK), catalog number: 1055/1 ] grown in 50 ml Falcon tubes
TransforMax^TMEPI300^TM Chemically competent E. coli cells (Lucigen, catalog number: C300C105 )
pKS diaCas9_sgRNA plasmid (Addgene, catalog number: 74923 )
BsaI-HF restriction endonuclease (New England Biolabs, catalog number: R0535S )
P1 buffer (QIAGEN, catalog number: 19051 )
β-mercaptoethanol (VWR, catalog number: 97064-588 )
Alkaline lysis (P2) buffer (QIAGEN, catalog number: 19052 )
Neutralization (P3) buffer (QIAGEN, catalog number: 19053 )
Isopropanol (Sigma-Aldrich, catalog number: 190764 )
95% (v/v) EtOH (Commercial Alcohols, catalog number: P016EA95 )
T4 DNA ligase with buffer (New England Biolabs, catalog number: M0202S )
2-Propanol-205 (Caledon Laboratories, catalog number: 8601-7-40 )
BSA (Sigma-Aldrich, catalog number: A2058 )
BsaI (New England Biolabs, catalog number: R0535S )
EZ-10 spin column Miniprep Kit (Bio Basic, catalog number: BS614 )
EZ-10 spin column PCR product purification kit (Bio Basic, catalog number: BS363 )
Bacto agar (BD, Bacto^TM, catalog number: 214030 )
Agar A (Bio Basic, catalog number: FB0010 )
Yeast extract (BioShop, catalog number: YEX401 )
Bacteriological tryptone (BioShop, catalog number: TRP402 )
Sodium chloride (NaCl) (Fisher Scientific, Fisher Chemical, catalog number: S271-500 )
AmpliTaq 360 DNA polymerase, 10x buffer, and 25 mM Magnesium Chloride (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4398828 )
Zeocin^TM (InvivoGen, catalog number: ant-zn-5p )
Ampicillin (BioShop, catalog number: AMP201.100 )
Gentamicin sulfate (Bio Basic, catalog number: GB0217 )
D-Glucose (BioShop, catalog number: GLU501.1 )
Zymolyase 20 T (BioShop, catalog number: ZYM001.1 )
Tris (Fisher Scientific, Fisher BioReagents, catalog number: BP152-5 )
Glycerol (Fisher Scientific, Fisher Chemical, catalog number: G33-1 )
Sodium sulfate (Na₂SO₄) (Fisher Scientific, Fisher Chemical, catalog number: S421-300LB )
Potassium chloride (KCl) (Merck, EMD Millipore, catalog number: PX1405-1 )
Sodium bicarbonate (NaHCO₃) (Merck, EMD Millipore, catalog number: SX0320-1 )
Potassium bromide (KBr) (The Science Company, catalog number: NC-2136 )
Boric acid (H₃BO₃) (BioShop, catalog number: BOR001.1 )
Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
Magnesium chloride hexahydrate (MgCl₂•6H₂O) (BioShop, catalog number: MAG510.1 )
Calcium chloride dihydrate (CaCl₂•2H₂O) (Fisher Scientific, catalog number: C79-3 )
Strontium chloride hexahydrate (SrCl₂•6H₂O) (Sigma-Aldrich, catalog number: 255521 )
Sodium nitrate (NaNO₃) (Fisher Scientific, catalog number: S343-500 )
Sodium phosphate monobasic monohydrate (NaH₂PO₄•H₂O) (Sigma-Aldrich, catalog number: S9638 )
Iron(III) chloride hexahydrate (FeCl₃•6H₂O) (Sigma-Aldrich, catalog number: 236489 )
Na₂EDTA•2H₂O (Bio Basic, catalog number: EB0185 )
Copper (II) sulfate pentahydrate (CuSO₄•5H₂O) (Bio Basic, catalog number: CDB0063 )
Sodium molybdate dihydrate (Na₂MoO₄•2H₂O) (Avantor Performance Materials, catalog number: 3764-01 )
Zinc sulfate heptahydrate (ZnSO₄•7H₂O) (Alfa Aesar, catalog number: A12915-36 )
Cobalt (II) chloride hexahydrate (CoCl₂•6H₂O) (Sigma-Aldrich, catalog number: C3169 )
Manganese(II) chloride tetrahydrate (MnCl₂•4H₂O) (BioShop, catalog number: MAN222 )
Selenous acid (H₂SeO₃) (Sigma-Aldrich, catalog number: 229857 )
Nickel(II) sulfate (NiSO₄) (Sigma-Aldrich, catalog number: 656895 )
Sodium orthovanadate (Na₃VO₄) (BioShop, catalog number: SPP310 )
Potassium chromate (K₂CrO₄) (Sigma-Aldrich, catalog number: 216615 )
Thiamine-HCl (Sigma-Aldrich, catalog number: 47858 )
Biotin (Sigma-Aldrich, catalog number: B4639 )
Cyanocobalamin (Sigma-Aldrich, catalog number: C3607 )
Zymogen solution (see Recipes)
Lysis buffer for P. tricornutum cells (see Recipes)
LB medium (see Recipes)
SOC media (see Recipes)
LB agar plates (see Recipes) containing 100 μg ml^-1 ampicillin or 100 μg ml^-1ampicillin with 40 μg ml^-1gentamicin
50% L1, 5% LB, 1% agar plate (1 L) (see Recipes)
L1 media (see Recipes)
L1 agar plates (see Recipes) containing 50 μg ml^-1 Zeocin^TM
2x Aquil salt (see Recipes)
NP stock (see Recipes)
1,000x L1 trace metals (see Recipes)
Vitamin solution (see Recipes)

Equipment

Qubit^® 2.0 Fluorometer (Thermo Fisher Scientific, Invitrogen^TM, model: Qubit^® 2.0 , catalog number: Q32866)
Heat block (VWR, catalog number: 13259-030 )
Water bath (Fisher Scientific, model: IC 2100 )
Micropipettes (PIPETMAN, variable volume)
Binocular Microscope (Leitz Wetzlar, model: Labolux12 )
Hemocytometer
Table top centrifuge (Hettich Lab Technology, catalog number: 2004-01 )
Centrifuge (Beckman Coulter, model: Avanti^® J-26 XPI , catalog number: 393127)
PCR thermal cycler (Bio-Rad Laboratories, model: T100^TM, catalog number: 1861096 )
UV-visible spectrophotometer (Biochrom, model: ULTROSPEC 2100^® , catalog number: 80-2112-21)
Incubator shaker (Thermo Fisher Scientific, model: Large Incubated and Refrigerated, catalog number: SHKE5000-7 )
Vortex
Autoclave
Refrigerator

Procedure

Designing Cas9-sgRNA targeting the urease gene
1. Obtain the pKSconj diaCas9 or diaTevCas9 (pPtGE34 or pPtGE35) plasmid (Figure 1) from Addgene (107932 or 107999).
  
  Figure 1. Plasmid map for pKSconj. diaCas9/diaTevCas9 (pPtGE34/pPtGE35)
2. Download the P. tricornutum urease gene sequence. NCBI Gene ID: 7194881 (PHATRDRAFT_29702).
3. Format the sequence in FASTA format with a sequence identifier on line 1 preceded by a > followed by the nucleotide sequence.
4. Use a custom Perl script to search for Cas9 and TevCas9 gRNA target sites in the urease gene, ~15 bp downstream of a 5’-CNNNG-3’ I-TevI cleavage site (see Supplemental files). A typical TevCas9 target site is illustrated in Figure 2.
  
  Figure 2. Illustration of TevCas9 target site with predicted deletion size
5. Select four target sites from the list of predicted sites for the P. trincornutum urease gene. Order an oligonucleotide corresponding to the predicted gRNA binding site (underlined sequence in Table 1), and the complementary oligonucleotide. Design the oligonucleotides with top strand 5’-TCGA-3’ and bottom strand 3’-CAAA-5’ BsaI restriction cut site overhangs to facilitate cloning into the Cas9 and TevCas9 vectors (Table 1).
  
  Table 1. Sequences of gRNAs targeting the urease gene in P. tricornutum
Cloning gRNAs into pKSconj diaCas9 and pKSconj diaTevCas9 plasmids
1. Phosphorylate and anneal the ordered oligos to generate gRNA cloning inserts. Prepare a 50 μl gRNA phosphorylation reaction (Table 2) in a 0.2 ml PCR tube. Place the reaction in a thermocycler and incubate at 37 °C for 30 min and then denature at 95 °C for 5 min. Next, slowly cool to 50 °C at a rate of -1 °C min^-1 to allow annealing of gRNA top and bottom strands.
  
  Table 2. Reagents for gRNA phosphorylation
2. Clone gRNAs into the pKSconj diaCas9 and pKSconj diaTevCas9 plasmids using Golden Gate assembly (Table 3). Place the reaction in a thermocycler and run the protocol as described in Table 4.
  
  Table 3. Reagents for Golden Gate assembly
  
  Table 4. Thermocycler setting for Golden Gate assembly
3. Transform the product from the Golden Gate reaction into Epi300 E. coli cells using heat shock as follows: For one reaction, mix 3 μl of Golden Gate assembly product with 30 μl of Epi300 E. coli cells. Incubate the reaction on ice for 30 min and heat shock at 42 °C for 45 sec, then incubate on ice for 2 min. Add 400 μl of SOC to the reaction and recover cells at 37 °C with shaking at 225 RPM for 90 min. Plate 150 μl of recovered cells onto LB + Ampicillin media and incubate at 37 °C for 16 h/overnight.
4. The following day, inoculate 1 to 5 colonies into separate glass test tubes containing 3 ml of LB + Ampicillin and incubate at 37 °C with shaking at 225 RPM for 16 h/overnight.
5. After overnight growth, take 3 ml of overnight culture to isolate plasmid DNA (Bio Basic Inc. plasmid miniprep kit). Send isolated plasmid DNA for Sanger sequencing at the London Regional Genomics Centre using a sequencing primer upstream of the gRNA insertion site to identify a plasmid containing a properly cloned gRNA.
6. Transform the properly cloned gRNA plasmid into Epi300 E. coli containing the pTA-Mob (Strand et al., 2014) plasmid, obtained from Dr. Rahmi Lale, using the heat shock protocol described in Step 3 with 1 μl of plasmid and 30 μl of E. coli cells.
Transfer of plasmid into P. tricornutum via conjugation from E. coli
Note: This protocol was adapted from a published protocol (Karas et al., 2015).
1. P. tricornutum growth conditions: 18 °C under cool white fluorescent light (75 μE m^-2 sec^-1) with a photoperiod of 16 h light and 8 h dark in 50 ml Falcon tubes at the Biotron Experimental Climate Change Research Centre at Western University.
2. Briefly, adjust 250 μl of P. tricornutum liquid culture to 1.0 x 10⁸ cells ml^-1, plate onto a 50% L1, 1% agar plate, and grow for 4 days.
3. Add 2 ml of L1 media to the plate, scrape and collect cells into a 1.5 ml Eppendorf tube.
4. Count the cells using a hemocytometer and adjust the concentration to 5 x 10⁸ cells ml^-1.
5. Grow 3 ml E. coli cultures containing the diaCas9 or diaTevCas9 plasmids with gRNAs at 37 °C overnight. Dilute 1 ml of overnight culture to 50 ml of LB with 100 μg ml^-1 ampicillin and 40 μg ml^-1 gentamicin and grow the culture to an OD₆₀₀ of 0.8-1.0.
6. Centrifuge the culture for 10 min at 3,000 x g in a 50 ml Falcon tube.
7. Discard the supernatant and resuspend the pellet in 500 μl SOC media.
8. Mix 200 μl of P. tricornutum from Step 4 with 200 μl of E. coli from Step 7 and plate them onto a 50% L1, 5% LB, 1% agar plate.
9. Incubate the plate for 90 min at 30 °C, then for 2 days at 18 °C with light.
10. After 2 days, add 1.5 ml of L1 media to the plate, scrape and collect cells in a 1.5 ml Eppendorf tube.
11. Plate 500 μl of the scraped cells onto a 50% L1, 1% agar plate containing 50 μg ml^-1 Zeocin^TM.
12. Parafilm the plate and incubate the plate for 14 days at 18 °C with light. A typical conjugation experiment should yield 1.0 x 10² - 1.3 x 10⁴ total exconjugants.
Screening for urease knockouts in P. tricornutum induced by Cas9 and TevCas9 (see Figure 3 for an overview of screening procedure) - T7 Endonuclease I assay

Figure 3. Overview of screening procedure for P. tricornutum urease knockouts. pKSconj diaCas9 or pKSconj diaTevCas9 plasmid is delivered to P. tricornutum by E. coli. Screen the exconjugants with T7EI assay. Isolate sub-clones from exconjugants and perform phenotypic screen by plating sub-clones onto urea and nitrate plates. Verify the deletion length by Sanger Sequencing.
1. After 14 days, randomly select ten P. tricornutum exconjugants and streak them onto an L1 + Zeocin^TM plate and grow for 7 days.
2. Scrape 50% of the cells from one streak and place it into 3 ml of L1 media with 50 μg ml^-1 Zeocin^TM. Suspend the other 50% of cells in 100 μl TE (pH 8.0) buffer, flash freeze at -80 °C for 15 min, and heat lyse at 95 °C for 5 min.
3. Use the lysed P. tricornutum exconjugant cells as the template for PCR amplification of the gRNA target site using AmpliTaq DNA polymerase. Design the PCR so the product will be ~500 bp using primers located ~250 bp upstream and downstream of the gRNA target site. Analyze the PCR products on a 1% agarose gel to confirm the size and presence of the PCR product.
4. Denature the PCR product at 95 °C for 5 min and slowly cool to 50 °C and then freeze the sample at -20 °C for 2 min to generate heteroduplexes of edited and unedited target sites.
5. Use the PCR product as the substrate for a T7EI assay (Table 5). Incubate the reaction at 37 °C for 15 min and analyze the digested product on a 1% agarose gel. PCR products from exconjugant that yield two bands on the agarose gel (500 bp and 250 bp) contains editing at the target site by Cas9 or TevCas9. An example of the T7EI assay gel is shown in Figure 4.
  
  Table 5. Reagents for T7EI assay
  
  Figure 4. Example analysis of Cas9 activity by T7EI assay. P. tricornutum colonies were resuspended in TE (pH 8.0) buffer followed by flash freezing at -80 °C for 15 min followed by cell lysis at 95 °C for 12 min. The urease gene target site was PCR amplified. PCR product was denatured at 95 °C and cooled down to 50 °C to allow random reannealing. T7E1 assay was performed using the randomly annealed PCR product as template and incubated at 37 °C for 15 min with T7EI and NEB 2 buffer. Products were loaded onto a 1% agarose gel at 100 V for 40 min. Asterisks (*) denote colonies positive for editing. The numbers (11.12, 11.13….) stand for different colonies.
6. Dilute the liquid cultures from Step D2 of P. tricornutum exconjugants that were identified to have editing to 10^-4 and plate onto L1+ Zeocin^TM plates and grow for 10-14 days to obtain sub-clones.
Phenotypic screening of urease knockouts (see Figure 5 for an example of the phenotypic screen)
1. Streak sub-clones first onto L1 + Zeocin^TM plates containing 2.1 mM urea with a pipette tip, use the same tip to streak the sub-clone onto an L1 + Zeocin^TM plate containing 4.2 mM nitrate and grow for 5 days. Sub-clones that grow on L1 + Zeocin^TM with nitrate but not on L1 + Zeocin^TM with urea are urease knockouts.
  
  Figure 5. Example phenotypic screen of P. tricornutum sub-clones on urea and nitrate plates. Dilutions of Cas9 and TevCas9 sub-clones were spot plated onto L1 with nitrate and L1 with urea plates.
2. PCR amplify the target site, as in Steps D2-D3, from sub-clones identified as urease knockouts and send the PCR product for Sanger sequencing.
Curing of the pKSconj diaCas9 and diaTevCas9 plasmids from urease knockout sub-clones
1. Inoculate identified urease knockout sub-clones in 3 ml of L1 media without Zeocin^TM and grow for 7 days.
2. After 7 days, dilute (10^-4) and plate the liquid cultures onto L1 plates and grow for 7 days to obtain single colonies.
3. Randomly select 10 colonies and streak them onto L1 and L1 + Zeocin^TM plates and grow for 5 days. Growth on L1 plates but not on L1 + Zeocin^TMplates indicates colonies that are successfully cured of the plasmid.

Data analysis

Ten exconjugants of P. tricornutum containing the pKSconj diaCas9 or diaTevCas9 plasmid with different gRNAs were randomly selected for T7EI analysis.
From those, 5 sub-clones were randomly selected for phenotypic screening.
To determine the removal of the plasmid, 10 colonies were randomly selected for Zeocin sensitivity screening.

Notes

Multiple gRNAs may need to be designed as the activity of Cas9 and TevCas9 varies when using different gRNAs.
The number of edited colonies may vary, more exconjugants may have to be selected to identify edited clones and obtain more accurate editing efficiencies.

Recipes

Zymolyase solution
200 mg zymolyase 20 T (USB)
9 ml dH₂O
1 ml 1 M Tris pH 7.5
10 ml 50% glycerol
Lysis buffer for P. tricornutum cells (1 L)
10 ml 1 M Tris pH 8.0
2 ml 0.5 M EDTA
988 ml dH₂O
LB medium (1 L)
10 g tryptone
5 g yeast extract
10 g NaCl
1 L dH₂O
Sterilize by autoclave at 121 °C with 1 atmosphere pressure for 15 min
SOC media (1 L)
20 g tryptone
5 g yeast extract
0.5 g NaCl
10 ml 250 mM KCl
5 ml 2 M MgCl
20 ml 1 M glucose
900 ml dH₂O
LB agar plates (containing 100 μg ml^-1 ampicillin or 100 μg ml^-1 ampicillin with 40 μg ml^-1 gentamicin) (1 L)
10 g tryptone
5 g yeast extract
10 g NaCl
15 g Agar
0.1 g ampicillin or 0.1 g ampicillin with 0.4 g gentamicin
1 L dH₂O
50% L1, 5% LB, 1% agar plate (1 L)
500 ml L1
50 ml LB
10 g bacto agar
L1 media (1 L)
500 ml 2x Aquil salt
2 ml NP stock
500 μl vitamin solution
1 ml 1,000x L1 trace metals
497.5 ml dH₂O
Sterilize by vacuum filtration with Nalgene^TM filters
L1 agar containing 50 μg ml^-1 Zeocin^TM (1 L)
500 ml L1 media
10 g bacto agar
0.5 g ZeocinTM
500 ml dH₂O
2x Aquil salt (1 L)
24.5 g NaCl
4.09 g Na₂SO₄
0.7 g KCl
0.2 g NaHCO₃
0.1 KBr
0.03 g H₃BO₃
0.003 g NaF
11.1 g MgCl₂•6H₂O
1.54 g CaCl₂•2H₂O
0.017 g SrCl₂•6H₂O
NP stock (100 ml)
37.5 g NaNO₃
2.5 g NaH₂PO₄•H₂O
1,000x L1 trace metals (1 L)
3.15 g FeCl₃•6H₂O
4.36 g Na₂EDTA•2H₂O
0.25 ml CuSO₄•5H₂O (9.8 g L^-1 dH₂O)
3.0 ml Na₂MoO₄•2H₂O (6.3 g L^-1 dH₂O)
1.0 ml ZnSO₄•7H₂O (22.0 g L^-1 dH₂O)
1.0 ml CoCl₂•6H₂O (10.0 g L^-1 dH₂O)
1.0 ml MnCl_2•4H₂O (180.0 g L^-1 dH₂O)
1.0 ml H₂SeO₃ (1.3 g L^-1 dH₂O)
1.0 ml NiSO₄•6H₂O (2.7 g L^-1 dH₂O)
1.0 ml Na₃VO₄ (1.84 g L^-1 dH₂O)
1.0 ml K₂CrO₄ (1.94 g L^-1 dH₂O)
Vitamin solution (1 L)
200 mg Thiamine-HCl
10 ml of 0.1 g L^-1 biotin
1 ml of 1 g L^-1 cyanocobalamin

Acknowledgments

This work was supported by Designer Microbes Inc., an NSERC ENGAGE Grant to D.R.E. and Designer Microbes Inc. (EGP/486420-2015), an Ontario Centres of Excellence VIP-1 Grant to Designer Microbes Inc. (OCE-VIP-1/23879), and NSERC Discovery Grant to D.R.E. (RGPIN-2015-04800).
This protocol was developed from the following published paper: Slattery et al. (2018).

Competing interests

The authors declare no conflicts of interests.

References

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