May 31, 2023
Prime Editing in Physcomitrium patens
- Pierre-François Perroud1,
- Anouchka Guyon-Debast 1,
- Josep M Casacuberta2,
- Wyatt Paul3,
- Jean-Philippe Pichon3,
- David Comeau3,
- Fabien Nogué1
- 1Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB), 78000, Versailles, France.;
- 2Centre for Research in Agricultural Genomics CSIC-IRTA-UAB-UB, Campus UAB, Edifici CRAG, Bellaterra, 08193 Barcelona, Spain;
- 3Limagrain Europe, Centre de Recherche de Chappes, 63720 , Chappes, France
Protocol Citation: Pierre-François Perroud, Anouchka Guyon-Debast , Josep M Casacuberta, Wyatt Paul, Jean-Philippe Pichon, David Comeau, Fabien Nogué 2023. Prime Editing in Physcomitrium patens. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27r5qg1y/v1
Manuscript citation:
Perroud P-F., Guyon-Debast A., Casacuberta J.M., Paul W., Pichon J-P., Comeau D. and Nogué F. Prime Editing allows for routine predictable gene editing in Physcomitrium patens. Journal of Experimental Botany (Manuscript under revision)
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: March 28, 2023
Last Modified: May 31, 2023
Protocol Integer ID: 79519
Funders Acknowledgement:
Agence National de la Recherche
Grant ID: ANR-10-LABX-0040-SPS
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Abstract
PEG-mediated naked DNA protoplast transfection has been the standard transformation approach for more than thirty years in the moss Physcomitrium patens. The advent of the CRISPR-Cas technology has given a new opportunity for this approach in moss and other plants. Recently, the Prime Editing, a CRISPR-Cas9 based approach combining the efficiency of genome searching of the nCas9, its association with a Reverse Transcriptase and the encoding of desired edit in 3' of the guide RNA has allowed the specific edition of any base-pair in the vicinity of the Streptococcus pyogenes Cas9 PAM –NGG . We present here a complete procedure of Prime Editing in P. patens starting from the production of the optimal tissue to obtain protoplasts, followed by generation of protoplasts, their transfection using PEG-mediated naked DNA and their subsequent regeneration. Finally we describe two modes of selection based on the choice of edited gene target. The direct selection mode is dedicated to the selection of edits generating a loss of function of the PpAPT gene that can be selected directly after regeneration on 2-Fluoroadenine-containing medium. The indirect selection mode allows for the pre-selection of transfected protoplast with an antibiotic independently of the targeted gene, the screen for the proper edit being identified after genotyping of the selected plants.
Guidelines
The present protocol is based on different procedures previously published (mainly Schaefer et al 1991, Cove et al. (2009) and Charlot et al. 2022). It contains four distinct sections: tissue production, protoplast production, protoplast transformation, and plant regeneration and selection.
Materials
If not mentioned otherwise, solutions are made using ultra pure water type milliQ or equivalent and is referred as mQ-H2O hereafter.
I. Specific material
Cellophane disk
Cellophane disks can either be purchased as such or be cut into disk from a cellophane sheet or roll. In both case the disk diameter should be 8.5 cm, so they fit standard 9 cm Petri dish after re-hydratation. Autoclave sterilization can be performed either in mQ-water, in which case they can be laid on medium directly, or dry condition in which case they need to be re-hydrated with sterile mQ-water before use.
II. Stock solutions:
8.5% (w/v) D-mannitol
D-mannitol 85 g
mQ-H2O up to 1 l
Autoclave, store at room temperature
CaCl2 1M
CaCl2.6H2O 219 g
mQ-H2O up to 1 l
Autoclave, store at room temperature
Driselase 2%
Driselase powder 2g
Driselase Plant tissue culture testedMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8037
8.5% (w/v) D-mannitol up to 100 ml
Proceed as follow: add both components and stir the solution for 30 minutes at room temperature. Transfer unto 50 ml tube and centrifuge 20 minutes at 4500 g. Filter sterilize using a 0.22 μm filter the supernatant that should be clear light brown. Make 5 ml aliquot in sterile tubes and store at -20ºC. Thaw 30 minutes before use.
MMM buffer
MES 2 g (1%)
Mannitol 17 g (8.5%)
MgCl2.6H2O 610 mg (15 mM)
mQ-H2O 180 ml
pH 5.6 with KOH and up to 200 ml with mQ-H2O and autoclave, store at room temperature.
PEG transformation solution
Mannitol 7 g (0.38 M)
Ca(NO3)2(H2O)4 2.36g (0.1M)
PEG 4000 (Serva,Roth) 40 g (40 % w/v)
Tris pH=8 1ml of 1M Tris pH=8.0
mQ-H2O up to 100 ml
Filter sterilize using 0.22 μm filter and make of 3 ml aliquot before storage at -20ºC
Phosphate-1000X Stock solution
KH2PO4 25g (184 mM)
mQ-H2O up to 80 ml
pH 7 with KOH, up to 100 ml with ddH2O and autoclave, store at 4ºC.
TES 1000X (Hoagland's A-Z trace element solution):
| H3BO3 | 614 mg | |
| MnCl2.4H2O | 389 mg | |
| Al2(SO4)3.K2SO4.24H2O | 55 mg | |
| CoCl2.6H2O | 55 mg | |
| CuSO4.5H20 | 55 mg | |
| ZnSO4.7H2O | 55 mg | |
| KBr | 28 mg | |
| KI | 28 mg | |
| LiCl | 28 mg | |
| SnCl2.2H2O | 28 mg | |
| H2O | up to 1 l |
Autoclave, store at 4ºC.
III.Media
Solid PPNH4 medium
| A | B | C | |
| Quantity | Final Concentration | ||
| Ca(NO3)2 4H2O | 0.8 g | 4 mM | |
| MgSO4 7H2O | 0.25 g | 1 mM | |
| FeSO4 7H2O | 12.5 g | 45 μM | |
| TES 1000X | 1 ml | ||
| Phosphate-1000X | 1 ml | 18.4 mM | |
| Ammonium tartrate dibasic | 0.5 g | 2.7 mM | |
| Agar, Plant tissue culture | 7 g | 0,7% | |
| H2Odist. | up to 1l |
Autoclave, pour unto 9 ml Petri dishes and store at room temperature.
Solid PPNH4 medium + Mannitol + CaCl2
| A | B | C | |
| Quantity | Final Concentration | ||
| Ca(NO3)2 4H2O | 0.8 g | 4 mM | |
| MgSO4 7H2O | 0.25 g | 1 mM | |
| FeSO4 7H2O | 12.5 mg | 45 μM | |
| Micro-elements-1000X | 1 ml | ||
| Phosphate-1000X | 1 ml | 18.4 mM | |
| Ammonium tartrate dibasic | 0.5 g | 2.7 mM | |
| Mannitol | 66 g | 6.6 % | |
| CaCl2 | 6 ml | 6 mM | |
| Agar, Plant tissue culture | 7 g | 0,7% | |
| H2Odist. | up to 1l |
Autoclave, pour unto 9 ml Petri dishes and store at store at room temperature.
Alginate solution
| A | B | C | |
| Quantity | Final Concentration | ||
| Mannitol | 8.5 g | 8.5 % | |
| Alginate | 1.5 g | 1.5 % | |
| ddH20 | 100 ml |
Ensure that the alginate is mostlty dissolved by heating at 65°C. prior autoclaving. Then, autoclave and filter with a 0.22 μm filter and make 50 ml aliquot . Store at room temperature.
IV.Antibiotic stock solutions
G418 1000X
G418 300 mg
mQ-H2O 10 ml
Filter with a 0.22 μm filter and make 1 ml aliquot . Store at -20ºC
Hygromycin B 1000X
Hygromycin B 250 mg
mQ-H2O 10 ml
Filter with a 0.22 μm filter and make 1 ml aliquot . Store at -20ºC
2-Fluoroadenine (2-FA) 1000X (10 mM)
2-FA 15.3 mg
DMSO 10 ml
Store at -20ºC
Before start
The protocol described hereafter needs to be performed at room temperature (<22°C) if not mentioned otherwise and in a sterile environment, e.g. vertical or horizontal laminar flow hood. Standard in vitro plant culture precautions concerning sterility (e.g. surface cleaning, reagent filtering and autoclaving) are required. The different solutions used for this protocol can and should be prepped before starting the procedure so to not lose time during its execution. Note that two components of the transfection protocol, the driselase 2% and the PEG transformation solution, are stored at-20°C, therefore before starting the procedure, bring them back to room temperature. The small metallic material (spatula and twizzer) can be sterilized just before use by flaming or using hot glass or metallic beads. Finally, for an optimum protoplast regeneration and protonemata growth, Petri dish with vent must be used and they must be sealed with 3M Micropore tape (or equivalent) to allow normal gas exchange in the plate (no parafilm).
Using a binocular lens, check carefully the starting P. patens material for an eventual contamination (bacteria, fungus). The tissue must be green. An axenic culture is essential before performing any further experimental step.
10m
P. patens protonema culture
P. patens protonema culture
1w
1w
Collect the tissue either from a seven day old protonemal culture or from spot inocculum with mixed tissue (protonema and gametophore) in a sterile 50 ml tube containing 20 ml sterile mQ-water.
2m
Using an homogenizer, grind the tissue into 5- 15 cells small fragments and filtrate the ground material through a sterilized 140 µm sieve. Recover the fragment left on the sieve with a sterile spatula and transfer it in a new 50 ml tube containing sterile mQ-water, adapt the water volume to the amount of recovered tissue.
1m
Inoculate 2 ml of the freshly fragmented protonema per Petri dish poured with solid PpNH4 medium overlaid with a sterilized cellophane disk. Seal the plates with 3M Micropore tape.
1m
Transfer the plate(s) in a incubator for seven days in standard plant growth condition (Temperature 22°C, light conditions: 16h light/ 8h darkness, 70 µE.m-2.sec-1).
1w
To obtain an homogenous protonemal culture necessary for the protoplast production, the repetition of the step 1-5 twice. These repetitions allow for the elimination of any gametophore tissue that will not protoplast and allow for the amplification of the tissue. Typically, one seven-days old entrained protonemal plate can be used to inoculate eight new plates.
2w
P. patens protoplast production
P. patens protoplast production
2h
2h
Harvest and transfer rapidly the protonemal tissue from four 6-7 days old cultures plates from Step 6 into 15 ml of 8.5% mannitol. Add 5 ml of Driselase 2% from frozen stock for a final Driselase concentration of 0.5% and incubate 40 minutes at room temperature with occasional gentle plate swirling
.
Note
With this protocol, protoplast yield should be between half million to one million protoplasts per 9 cm Petri dish culture.
Filter the suspension through 80-100 µm sieve and let incubate at room temperature for 10 minutes.
Filter the suspension to 40-50 µm sieve and pour the flow-through (containing the isolated protoplasts) into a tube that can withstand low speed centrifugation (e.g. 50 ml conical tube).
Centrifuge 5 minutes at 150 x g to sediment the protoplasts.
Living protoplasts will sediment to form a dark green pellet. Discard the supernatant by tilting the tube and gently resuspended the pellet unto 20 ml of mannitol 8.5% supplemented with 10 mM CaCl2 final.
Centrifuge 5 minutes at 150 x g to sediment the protoplasts.
Living protoplasts will sediment to form a dark green pellet. Discard the supernatant by tilting the tube and gently resuspended the pellet unto 20 ml of mannitol 8.5% supplemented with 10 mM CaCl2 final. Evaluate the number of obtained protoplasts using a Malassez counting chamber or equivalent.
Centrifuge 5 minutes at 150 x g to sediment the protoplasts. Discard the supernatant by tilting the tube. Proceed directly to the transfection procedure.
P. patens protoplast transfection and plating
P. patens protoplast transfection and plating
In a 15 ml sterile tube, put 15 µg of total vector DNA necessary for the Prime Editing procedure resuspended in sterile mQ-water in a maximum of 30 µl volume. This DNA can be produced by standard PCR amplification or plasmid prep amplification. The two vectors vectors necessary are the expression vector for the Prime editor and the vector expressing the pegRNA specific to the target of choice. In addition a third vector containing an antibiotic resistance cassette must be added in case of indirect selection of transfectants.
1m
Add 300 µl of protoplast ressuspended in MMM buffer to the vector DNA. Protoplast concentration in the MMM buffer can be anywhere between 1.2x106 and 1.6x106 without affecting transformation efficiency with the volume ratio for each component presented in this protocol.
1m
Add 300 µl of PEG transformation solution (from frozen stock) and mix gently (do not vortex or pipet up and down the suspension) but thoroughly, to produce a visually uniform solution.
1m
Incubate the tube for 5 minutes in a 44°C water bath.
5m
Let the tube stand at room temperature for 10 minutes.
10m
The PEG transformation solution is toxic to the protoplasts. Dilute the transformation mix by adding, at one minute interval, five times 300 µl, and then five times 1 ml of mannitol 8.5% supplemented with 10 mM CaCl2 final (6.5 ml total). Mix gently by swirling the tube after each dilution step.
10m
Let the tube stand at room temperature for 30 minutes. At this stage protoplasts have been transformed and are ready to be plated.
To concentrate the protoplast suspension, centrifuge 5 minutes at 150 x g to sediment the protoplasts. Discard the supernatant and add up to 4 ml of fresh mannitol 8.5% supplemented with 6 mM CaCl2 final for one transformation tube.
5m
Add 4 ml of alginate solution to the protoplast suspension and mix gently by pipetting up and down.
30s
Pour readily 2 ml of mixture per 9 cm Petri dish filled of solid regeneration medium overlayed with sterile cellophane. Each standard transfection is spread on four 9 cm Petri dishes.
30s
Let the plates stand for 30 minutes to one hour in the laminar flow hood (covered and sealed) before transferring the plate to the incubator (Temperature 22°C, light conditions: 16h light/ 8h darkness, 70 µE.m-2.sec-1).
30m
Let the protoplasts grow for 6 to 7 days on the transformation plates. Observe the plates for the number of divided protoplasts since this is the actually number that count to evaluate transformation efficiency if necessary. After a week of growth, most of what can regenerate will have and you can identify them as plant with cells number between 2 and 15 cells
1w
Selection mode is dependent of the edited target. If the edited target can be selected positively, e.g. the non edited plant dies on the selection medium and the edited plant survives as it is the case for for endogenous gene PpAPT and the selection compound 2-Fluoroadenine (2-FA) use the Plant direct selection case. If the locus phenotype cannot be directly selected or generate an unknown phenotype used the Plant indirect selection case.
Plant direct selection 4 steps
To use when the gene PpAPT is targeted and yields apt- variant resistant to 2-FA.
Transfer the cellophane containing the regenerating protoplasts onto PpNH4 plate supplemented with 10 µM 2-FA and place the plates for at least a week in standard plant growth condition (Temperature 22°C, light conditions: 16h light/ 8h darkness, 70 µE.m-2.sec-1). During this step, only the plants that have the gene PpAPT disrupted will grow as 2-FA is lethal to the wild-type strain.
The selected plants are now ready for tissue amplification on standard PpNH4 medium and subsequent genotyping according to the specific target edition in the gene PpAPT.
1w
Plant indirect selection 2 steps
To use when the phenotype generated by the edit is not selectable directly or unknown
Transfer the cellophane containing the regenerating protoplasts onto solid PpNH4 medium plate supplemented with the appropriate antibiotic (for which resistance is present in the transformation vector). Incubate plates for at least a week in standard plant growth condition (Temperature 22°C, light conditions: 16h light/ 8h darkness, 70 µE.m-2.sec-1). During this step most of the plants should die, leaving 50 to 250 growing plants.
1w
Under a binocular lens, isolate individual antibiotic resistant plant using sterile twizzer and transfer them unto fresh PpNH4 medium and incubate plates for at least a two weeks in standard plant growth condition (Temperature 22°C, light conditions: 16h light/ 8h darkness, 70 µE.m-2.sec-1). At this point selected plant are ready for genotyping and identification of the edit event.
2w
Protocol references
Charlot F, Goudounet G, Nogué F, Perroud P-F (2022) Physcomitrium patens Protoplasting and Protoplast Transfection. In: Wang K, Zhang F (eds) Methods in molecular biology (Clifton, N.J.)-Protoplast Technologies. Springer Nature, pp 3–19
Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) The Moss
Physcomitrella patens. A Novel Model System for Plant Development and Genomic Studies. In 'Emerging Model Organisms, a Laboratory Manual'. (Eds C D.A. and G A.) pp. 69-104 Cold Spring Harbor Laboratory Press: New York
Schaefer DG, Zryd J-P, Knight CD, Cove DJ (1991) Stable transformation of the moss Physcomitrella patens. Mol. Gen. Genet. 226:418–424.