TITLE: Prime Editing in Physcomitrium patens
AUTHORS:  Pierre-François Perroud, Anouchka Guyon-Debast , Josep M Casacuberta, Wyatt Paul, Jean-Philippe Pichon, David Comeau, Fabien Nogué

[DESCRIPTION]
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.

[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).

[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. 

[STEPS]
1. 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. 
26. 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
SECTION: P. patens protonema culture
3. 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.
SECTION: P. patens protonema culture
2. 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. 
SECTION: P. patens protonema culture
4. 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.
SECTION: P. patens protonema culture
5.  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).
SECTION: P. patens protonema culture
6. 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.
SECTION: P. patens protoplast production
7. 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
.
 

SECTION: P. patens protoplast production
8. Filter the suspension through 80-100 µm sieve and let incubate at room temperature for 10 minutes.
SECTION: P. patens protoplast production
9. 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).
SECTION: P. patens protoplast production
10. Centrifuge 5 minutes at 150 x g to sediment the protoplasts.
SECTION: P. patens protoplast production
11. 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.
SECTION: P. patens protoplast production
12. Centrifuge 5 minutes at 150 x g to sediment the protoplasts.
SECTION: P. patens protoplast production
13. 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.
SECTION: P. patens protoplast production
14. Centrifuge 5 minutes at 150 x g to sediment the protoplasts. Discard the supernatant by tilting the tube. Proceed directly to the  transfection procedure.
SECTION: P. patens protoplast transfection and plating
15. 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. 
SECTION: P. patens protoplast transfection and plating
16. 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.
SECTION: P. patens protoplast transfection and plating
17. 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.
SECTION: P. patens protoplast transfection and plating
18. Incubate the tube for 5 minutes in a 44°C water bath.
SECTION: P. patens protoplast transfection and plating
19. Let the tube stand at room temperature for 10 minutes.
SECTION: P. patens protoplast transfection and plating
20. 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.
SECTION: P. patens protoplast transfection and plating
21. Let the tube stand at room temperature for 30 minutes. At this stage protoplasts have been transformed and are ready to be plated.
SECTION: P. patens protoplast transfection and plating
22. 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.
SECTION: P. patens protoplast transfection and plating
23. Add 4 ml of alginate solution to the protoplast suspension and mix gently by pipetting up and down. 
SECTION: P. patens protoplast transfection and plating
25. 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). 
SECTION: Transfected P. patens plant selection
27. 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.

SECTION: Transfected P. patens plant selection
29. 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.
SECTION: Transfected P. patens plant selection
31. 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.
SECTION: Transfected P. patens plant selection
30. 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.
SECTION: P. patens protoplast transfection and plating
24. 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.
SECTION: Transfected P. patens plant selection
28. 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.