A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti
- T. gondii ME49 wild type, ME49ΔKu80, Besnoitia besnoiti strain Lisbon 14
- Human foreskin fibroblasts, HFF (ATCC, Cat. # SCRC-1041)
- General cell culture equipment (e.g., T25 cell culture flasks (TPP, Cat. # 90026), cell scraper (TPP, Cat. # 99002), syringes (Henry Schein, Cat. # 9003311), 24G and 22G needles (Henke Sass Wolf, 4710005525 and 4710007030) , 96-well plates (TPP, Cat. # 92096), serological pipettes (Sarstedt, Cat . # 86.1253.001, 86.1254.001), 5 μm syringe filters (Henry Schein, Cat. # 9003311)
- 2 mm gap electroporation cuvettes (Biorad Gene Pulser Cuvette, Cat. # 165-2086)
- PCR tubes (Abgene, Cat. # AB1182)
- Forward and reverse primers (see below)
- Thermococcus kodakaraenis (KOD) DNA polymerase (Merck Millipore, Cat. # 71085)
- KOD polymerase generated PCR amplicons (see below)
- PCR purification kit (Promega Wizard SV Gel and PCR Clean-Up System, Cat. # A9282)
- Agarose gel
- GelRed nucleic acid stain (Biotium, Cat. # BI 41003)
- DNA Gel Loading Dye (Thermo Fisher, Cat. # R1151)
- Gene Ruler DNA TM Ladder Mix (Fermentas, Cat. # SM0333)
- 3 M sodium acetate in H2O, pH 5.2
- 100% and 70% EtOH in H2O
- Cas9 NLS (NEB EnGen Cas9 NLS, S. pyogenes, Cat. # M0646T)
- Chemically modified crRNA and tracrRNA
- p2854 DHFR-TS*  and HA-DHFR-TS*(unpublished) plasmids
- gDNA extraction kit (Thermo Fisher, Phire tissue direct PCR Master kit, Cat. # F-170S)
- Diagnostic primers
- Online tools:
- Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Cat. # D6429) supplemented with 10% heat inactivated fetal bovine serum (FBS, Bioswisstec, Cat. # S0613), 2 mM L-glutamine (Sigma, Cat. # G7513-100 ml) and 100 units/ml penicillin, 0.10 µg/ml streptomycin, 0.15 µg/ml amphotericin B (PSF, Gibco, Cat. # 15240062-100 ml).
- PBS: 0.137 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4, pH 7.4.
- Cytomix: 120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4 pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 5 mM MgCl2 in H2O.
- Adenosine 5’-triphosphate disodium salt (ATP): 100 mM in H2O, adjusted to pH 7.0 with KOH.
- Glutathione (GSH): 100 mM in H2O, pH to 7.0 with KOH.
- Annealing buffer: 30 mM HEPES, pH 7.5, 100 mM potassium acetate.
- Intracellular (IC)-buffer: 5 mM NaCl, 142 mM KCl, 1 mM MgCl2, 2 mM EGTA, 5.6 mM Glucose, 25 mM HEPES, pH to 7.2 with KOH.
- Pyrimethamine (Lubio Science Selleckchem, Cat. # S2006), 10 mM stock solution in DMSO.
- 5-Fluoro-2’-deoxyuridine (FUDR, Sigma, Cat. # F0503), 10 mM stock solution in H2O.
- Thermocycler (Biorad C1000 Touch, Thermal Cycler)
- Agarose Electrophoresis System (Biorad, PowerPac HC and gel chamber)
- Chemiluminescence Imager (Witec, Multi Wavelength Illuminator)
- Nanodrop (Thermo Scientific, NanoDrop One Microvolume UV-Vis Spectrophotometer)
- Electroporation system (Bio-Rad Gene Pulser X cell)
- Cooling centrifuge with 15 ml tube holders (Beckman Coulter) and Eppendorf tube holders (Eppendorf Centrifuge 5418R)
GOI sequence retrieval and crRNA design
1.Genomic target DNA sequences can be retrieved from ToxoDB (http://toxodb.org/toxo/).
2.Choose an optimal crRNA sequence using the online tool Eukaryotic Pathogen CRISPR guide RNA/DNA Design Tool http://grna.ctegd.uga.edu/.
3.For C-terminal tagging, choose a crRNA that is targeting a few basepairs downstream of the stop codon. The crRNA for N-terminal tagging should be chosen a few basepairs upstream of the start codon.
4.For gene knockout, design the 5′ and 3′ homology region upstream of the start codon respectively downstream of the stop codon. Both 3′ and 5′ homology regions should be around 30 bp (depicted as n). The KOD PCRs are performed on the DHFR-TS* vector with the following primers:
5.Assemble the following reagents. 8 × 50 μl KOD reactions are generally sufficient for one transfection (40 μg).
6.Mix reagents completely in PCR tubes and then transfer to the thermocycler.
7.Run the following cycling program, whereas conditions 2–4 are repeated 34 times:
8.Purify PCR products (e.g., the Promega Wizard SV Gel and PCR Clean-Up System)
9.Check the PCR product quality by agarose gel electrophoresis.
10.Determine the concentration of the PCR product by Nanodrop.
11.Precipitate 40 μg of the PCR product using 10% sodium acetate 3 M and 3× volumes of ice-cold 100% EtOH.
12.Incubate at −20°C for > 20 min.
13.Centrifuge at 16000 × g for 30 min at 4°C.
14.Discard the supernatant.
15.Wash the pellet by adding 1000 μl 70% ice-cold ethanol. Either store the pellet in 70% ethanol at −20°C or proceed directly for transfection.
16.Centrifuge the pellet at 16000 × g for 10 min at 4°C and discard the supernatant.
17.Air-dry the pellet for approximately 20 min in the laminar flow, and then dissolve in 100 μl cytomix supplemented with 2 mM ATP and 5 mM GSH.
Preparation of the RNP complex
18.Dissolve crRNA and tracrRNA in nuclease-free water each to 100 µM.
19.Dilute crRNA and tracrRNA in annealing buffer to 40 µM.
20.Mix crRNA and tracrRNA equimolar in a PCR tube.
21.Follow a temperature gradient protocol to anneal crRNA:tracrRNA (sgRNA formation, Fig. 4A). Start with 92°C for 2 min and gradually decrease the temperature over 30 min to RT.
22.Mix 5.8 µl IC buffer and 0.6 µl NEB Cas9 NLS.
23.Mix 4 µl of the cooled sgRNA with Cas9/IC-buffer and incubate at RT for at least 20 min, which results in the RNP complex formation (Fig. 4B).
Parasite transfection and drug selection
24.Grow parasites in a T25 cell culture flask on HFF for 3–4 d until big vacuoles and freshly egressed parasites are visible (Fig. 4C).
25.Scrape the infected HFF monolayers from the T25 flask and homogenize cells by syringe passage through 24 G and 22 G needles to fully release the parasites.
26.Filter the parasites with a 5 µm syringe filter into a falcon tube.
27.Centrifuge at 1000 × g, 5 min, 4°C and discard the supernatant.
28.Wash the parasites once in ice-cold PBS; use an aliquot for parasite count using a hemocytometer. One T25 flask typically results in 5 × 107 parasites.
29.Centrifuge at 1000 × g, 5 min, 4°C and remove the supernatant.
30.Resuspend the parasites in 300 µl cytomix supplemented with 2 mM ATP and 5 mM GSH.
31.Mix 100 µl KOD PCR product, 300 µl parasites, and the RNP complex and transfer to pre-cooled 2 mm electroporation cuvette.
32.Electroporate the parasites with 2 pulses of a pulse length of 0.3 ms in an interval of 5 s with 1.5 kV.
33.After electroporation, let the cuvette stand for 5 min at RT.
34.Add the transfected tachyzoites to a confluent T25 flask with HFF cells and let grow at 37°C with 5% CO2 in a humidified incubator.
35.24 h post-transfection, drug selection is initiated with 1 µM pyrimethamine for resistance to DHFR-TS* in T. gondii or 10 µM FUDR for B. besnoiti for selection of uprt knockout mutants.
36.After 3–4 d, drug-resistant parasites emerge.
37.Single clones are isolated by limiting dilution cloning.
38.Amplify single clones by passaging to get enough material for genomic DNA extraction and the assessment for successful integration of the selection marker by diagnostic PCR and Sanger sequencing.
TIDE analysis for efficiency control
39.Use the ME49 wild type strain instead of the ΔKU80 as only the wild type parasites have the capability of introducing indels that can be monitored by TIDE.
40.Transfection of the RNP is sufficient, as there is no need for a selection marker.
41.Perform a mock transfection with wild type parasites in absence of an RNP complex.
42.After 3–4 d, harvest the parasites (Steps 25–29).
43.Extract the genomic DNA.
44.Perform a standard PCR with genomic DNA as template from the mock and RNP transfection. Primers should be designed according to the instructions section in https://tide.deskgen.com/.
45.Purify the PCR products using e.g., the Promega Wizard SV Gel and PCR Clean-Up System.
46.Sequence the PCR products using standard sequencing reactions.
47.Enter the 20 nt crRNA sequence as well as the chromatogram (.ab1) files from the sequencing reaction into TIDE. The chromatogram from the mock transfection is entered under “Control Sample Chromatogram (.ab1)” and the Chromatogram from the RNP transfection under “Test Sample Chromatogram (.ab1)”.
48.Pressing the “Update View” button will display the transfection efficiency.
|2||The efficiency of single crRNAs can vary widely||Efficiencies vary based on the nucleotide compositions and secondary structures of sgRNAs||It might be necessary to design more than one crRNA if first trials are not successful|
|11,17||Arcing during electroporation||An excess of salts present in the transfection reaction might cause arcing and lead to reduced viability of the parasites and transfection efficiency||PCR products should therefore be precipitated and IC buffer volume should be minimal|
|48||The transfection efficiencies reached in the two parasites is close to the minimal detection limit of TIDE||TIDE is designed for transfection efficiencies of up to 30%, reached in human cell lines||To monitor efficiencies in parasites, the experiment should be repeated three times; similar efficiency outcomes indicate a correct result|
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