Generation of transgenic lines

CRISPR experiment design

1. Design your sgRNA (N₂₀ + NGG)

1. Copy sequence of the region where you want to design the guide into a sgRNA design website:
   1. Benchling
   2. http://grna.ctegd.uga.edu/
   3. Protospacer
2. Select the best sgRNA based on:
   1. Number of off targets (the lower the better).
   2. GC content (the closer to 50% the better).
   3. Avoid tracks of As, Ts or ATs in the guide.
   4. Guide specificity: Blast in PlasmoDB – to see if it binds to other sites in the genome.
      1. Tools → Blast → Genome → Pf 3D7 → Copy guide sequence
      2. The higher the difference is between the first and second blast hit the better.
      3. Off-targets are only a concern if the 12 bp next to the NGG are identical to the target.
   5. Guide self-complementarity: using the tool “Oligocalculator” (http://biotools.nubic.northwestern.edu/OligoCalc.html) (select guides that don’t form hairpins, …)
3. Once a sgRNA has been selected, synthesize two oligonucleotides to clone the guide in a plasmid using the In Fusion system:

2. Design your two homology regions

1. Normally each HR should have between 300-500bp.
2. At least one HR should be near the sgRNA (ideally <20 bp, maximum 30 bp). The other can be far, but not more than 3000 bp (if this is the case, you could use 2 sgRNA at the same time).
3. Try to avoid introns and areas with large (T)n, (A)n or (TA)n tracks as they complicate the process later on.
4. The HR can include the sgRNA, but if this is the case this part of the HR should be recodonized (important to change the PAM!) so that it is not cut by the Cas9 after correct editing.

IN FUSION LIGATION (sgRNA)

Notes

• Based on protocols provided by José-Juan Lopez Rubio.

Protocol

1. Linearize the vector by restriction enzyme digestion (BtgZI or BbsI, depends on which plasmid you are using) and purify it.

2. Annealing of the sgRNA oligos:

1. Resuspend the stock oligos at 50 µM with H2O.
2. Mix 10 µl of each oligo + 2.2 µl NEB2 buffer
3. Heat 400-500 ml of H2O to 95ºC in the microwave. Place the Eppendorf with the annealing mixture floating in the heated water and leave it until it reaches room temperature (25ºC approx). Control the temperature with a mercury thermometer.
4. Dilute oligos to 0.5 µM (dilution 1:50) in cold 5 mM Tris pH=8 buffer*. After the annealing they will be 25 µM, so a 1:50 dilution needs to be done.
5. Keep on ice.

* 5 mM Tris pH=8: dilute 1:200 from 1M Tris-HCl pH=8 (6.057g Trizma base in 50ml H2O, adjust pH with HCl)

3. Prepare a 5µl reaction:

* Use 100 ng of linearized vector. It should fit in 2.5 µl, so you should have a minimum concentration of 40 ng/µl. Otherwise, increase the reaction volume or concentrate the sample using a SpeedVac.

4. Incubate the reaction for 15 min at 50 °C in a thermocycler.

5. Keep on ice.

6. Use 2.5 µl to transform DH5α competent cells.

TRANSFECTION

PLASMID PREPARATION

Notes:

• Do Maxipreps of the plasmids you want to transfect. We use the Qiagen EndoFree Plasmid Maxi kit. Before transfecting, check by sequencing that your plasmids are ok.
• If you use a classical homologous recombination strategy, transfect 100 µg of circular plasmid.
• If you use a CRISPR/Cas9 strategy, transfect ~12 µg of linear donor plasmid and 60 µg of circular Cas9 plasmid. The plasmid with the donor DNA should be linearized using an enzyme that cuts as far away as possible from the homology regions and donor DNA (e.g. ScaI, XmnI).
• Remember to leave the phenol:chloroform:isoamilic for 1h at RT before using it.
• Precipitating the plasmids with ethanol is necessary to make them sterile andavoid the risk of culture contamination.
• Prepare NaAc 3M pH=5.2* and Cytomix** if necessary, using the followingrecipes:

• NaAc 3M pH=5.2 For 200 mL:

       49.21 g NaAc  anhydrous Add mili Q water to 20 0mL Adjust pH 5.2 with acetic acid

      Autoclave and store at room temperature

• Cytomix

        [120 mM KCl, 0.15 mM CaCl₂, 2 mM EGTA, 5 mM MgCl₂, 10 mM

        K₂HPO₄/KH₂HPO₄ 25 mM HEPES pH 7.6]

        For 250 mL:

       15 mL 2 M KCl

       18.75 µL 2 M CaCl₂

       1.25 mL 1 M MgCl₂

       2.5 mL 1 M K₂HPO₄/KH₂HPO₄ pH 7.6 25 mL 250 mM HEPES/20 mM EGTA

       Adjust pH 7.6 with 1 M KOH (~500-650 µL) Filter-sterilise and store 4ºC

Plasmid linearization and purification:

1. Quantify maxiprep.

2. Prepare a digestion reaction with a final volume of 50µl that includes 15 µg of plasmid from maxiprep.

3. Leave to digest at 37ºC at least 3h.

4. Run the linearized DNA (0.2 µl) on a gel to check that it has the expected size.

5. Purify the DNA from the reaction:

1. Adjust the volume of the digestion to 200 µl using H2Omq
2. Add one volume (200 µl) of phenol chloroform (stored in the fridge, should be kept at room temperature for at least an hour before use).
3. Mix thoroughly
4. Centrifuge at maximum speed for 5 min at RT.
5. Recover the upper phase (~160-170 µl) into another tube.

6. You can expect to end up having ~12 µg of linear plasmid (no need to quantify it).
 

Plasmid precipitation:

1. Quantify the concentration of themaxiprep.

1. For linear plasmids: perform first the “Plasmid linearization andpurification” protocol. Use all of the linearized DNA (~12µg).
2. For circular plasmids: start with 60 µg of plasmid from maxiprep.

2. Add H2O to a final volume of 270 µl

3. Add 30 µl of NaAc 3 M pH=5.2 (Dil1:10)

4. Add 750 µl of cold (-20ºC) EtOH (2.5x volume)

5. Invert a few times (you can see the precipitated DNA if working with 60 µg).

6. Leave at -20ºC for at least 24 h until the day of transfection

Plasmid resuspension:

You can do the next steps just before transfection or some days before and freeze the resuspended plasmids at -20ºC until transfection.

1. Centrifuge 30 min at 4ºC full speed (small centrifuge).

2. From here on, work in the culture room biosafety cabinet under sterile conditions.

3. Remove the supernatant (first with a P1000 pipette and then finish removing with a P200).

4. Resuspend the pellet in 1 ml of sterile EtOH 70% (at -20ºC) by lightly tapping the Eppendorf.

5. Centrifuge for 10 min at 4ºC (smallcentrifuge).

6. Remove the supernatant with a P1000Pipette.

7. Give it a quick spin and then remove the remaining supernatant with a p200 pipette.

8. To dry the pellet, leave the tube with the lid open for 5 min in sterile conditions until you see no liquid drops.

9. Resuspend in 30 µl sterile TE Buffer. If your strategy requires the transfection of 2 plasmids, resuspend both of them in a total volume of 30µl TE. At this step, 2 µl can be taken to be quantified or run on a gel.

10. TE-resuspended plasmids can be directly used for transfection or stored at -20ºC until transfection.
 

CULTURE PREPARATION

1. Previously thaw a parasite stock and maintain it for a few cycles.

2. Prepare a healthy growing culture with 5-8% of rings. Keep in mind that you will use 6,7ml of culture at 3% haematocrit (containing 200µl of RBC) for each transfection.

3. On the day of transfection, perform a sorbitol synchronization and allow the culture to recover ~1h at 37ºC before transfection.

TRANSFECTION

1. Pipette your culture up and down until RBCs are homogeneously resuspended and transfer 2/3 of the 10ml culture (6,7 ml of culture, containing ~200 µl of RBC) to a 15 ml tube.

2. Centrifuge for 5 min at 1500 rpm and remove the supernatant.

3. Add 370 µl of Cytomix (stored at 4ºC, no need to warm in the water bath) to the 30 µl of precipitated plasmid.

4. Add the 400 µl of Cytomix+plasmid to the RBC pellet and mix by pipetting up and down.

5. Transfer to an electroporation cuvette using a 1 ml serological pipette. Be careful not to make bubbles, and do not touch the metallic part of the cuvette!

6. Electroporate in a BioRad GenePulser Xcell (310V, 950uF capac, ∞Ω, 2mm cuvette) (constant time 12.7, V=307 V)

7. Recover the contents of the cuvette with a sterile fine tip plastic Pasteur pipette and transfer to a 15 ml tube.

8. Wash the cuvette twice with pre-warmed washing media and transfer it to the same tube.

9. Centrifuge for 5 min at 1500 rpm and remove the supernatant.

10. Resuspend with 12 ml of completemedia.

11. Add fresh blood RBCs to a total of 300µl. The electroporated sample already has 200 µl of RBC; therefore, only 100 µl of additional RBCs are required. But since during the electroporation some RBC may be lysed, it is recommendable to add 150 µl (i.e., 300 µl RBC 50%).

DRUG SELECTION

Available drugs:

• WR99210: we routinely use it for positive selection.
• Blasticidin
• Ancotil: in some cases, we use it for negative selection.

WR99210

• If you use a strategy in which the resistance marker is integrated in the genome and NOT using CRISPR/Cas9, use WR99210 selection until parasites start to grow well. Then, do 2-3 cycles off-on drug: maintain parasites without selection for 2 weeks and afterwards add the drug again until parasites start to grow well again.
• If you use a marker-free CRISPR-Cas9 strategy, keep parasites under WR99210 selection for only 4 days.
• If you use a CRISPR-Cas9 strategy in which the marker is integrated in the genome, keep parasites under WR99210 selection until they start to grow and you have sufficient material for genetic analysis.

1. Prepare WR99210:
   1. WR99210: 25 µl aliquots of 20 mM WR99219 are frozen at -80ºC.
   2. Prepare WR99210 solution at 20 µM (dilute 1: 1000) by mixing 25 µl in 25 ml of washing media.
   3. Filter the solution with a syringefilter.
   4. Store at 4ºC for up to 1 month.
2. The day after transfection (12-24 h later, when most parasites are at the trophozoite stage), add the drug (dilution 1:2000 of the 20 µM stock, eg. add 25 µl in 50 mL of complete media if you have several cultures). To do so:
   1. Centrifuge culture and remove supernatant.
   2. Resuspend the culture with complete media + WR99210.
3. For the next 3 days change the media + WR99210.
4. If using a marker-free strategy, after the 4 days of selection, wash the culture to eliminate the remaining drug and maintain without drug for the next few weeks. Otherwise, maintain with WR99210 until parasites start growing.
5. To maintain transfections until parasites start to appear (with or without drug):
   1. Change media every two/three days
   2. Dilute 1:2 once a week with fresh blood. The first week after transfection, you can dilute 1:2 and transfer into a 4ml plate (discard part of the culture).
   3. Make smears regularly to check forparasites.
6. As soon as you can, freeze parasitestocks.
7. As soon as you can, extract gDNA from transgenic parasites to check if they are correctly edited.