CRISPR-Cas12a ribonucleoprotein-mediated gene editing in the plant pathogenic fungus Magnaporthe oryzae

Summary Gene replacements through homologous recombination (HR) have been extensively used for functional genomic studies. However, the general efficiency of HR repair can be low in filamentous fungi and the process laborious. Here, we provide a detailed protocol for efficient gene editing by inserting donor DNA into a region of interest following Cas12a ribonucleoprotein (RNP)-mediated DNA double-strand break. We demonstrate this protocol using Magnaporthe oryzae (synonym of Pyricularia oryzae), a model plant pathogenic fungus that is used to study plant-fungal interactions. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021).


MATERIALS AND EQUIPMENT Prepare medium and stock solution
Timing: 1 week Oatmeal agar (OTA) Dissolve 72.5 g commercial OTA powder in 1 L of distilled water, mix well. Heat to near boiling using a microwave for 1-2 min. Autoclave at 121 C for 20 min in liquid cycle. Pour the sterilized OTA in a 100 3 15 mm petri dish after autoclave, store at room temperature ($28 C). The plate can be stored for up to 2 months.
CRITICAL: It is very important to preheat the OTA before autoclave steps.
Alternatives: Microwave can be replaced with a water bath for pre-heating.

Vector usage
We amplified the coding sequence for hygromycin, g418 and Bialaphos from the vectors pFGL821, pFGL921 and pBV9/pSM324 respectively. Additional details can be found in the Note of step 9. Other vectors or templates can additionally be used to amplify other genes of interest.

STEP-BY-STEP METHOD DETAILS
Design oligos for LbCas12a guide RNA

Timing: 1 week
This step is required to design a guide RNA that will direct the Cas12a nuclease to a specific site for editing. To demonstrate CRISPR-Cas12a editing in M. oryzae, we mutated the trihydroxynaphthalene reductase encoding gene termed BUF1 (MGG_02252), which is involved in fungal melanin biosynthesis (Chumley and Valent, 1990). The buf1 deletion mutant showed buff color (i.e., tan/orange) compared with the gray/black mycelial color in the wild-type strain. In this step, we describe the procedure to design the Cas12a guide RNA targeting BUF1.
1. Download the sequence of gene of interest from FungiDB (https://fungidb.org/fungidb/app). For the BUF1 gene, download the sequence for the gene MGG_02252. 2. Search for the LbCas12a PAM sequences (5 0 -TTTV-3 0 ) in your gene of interest. Select a 23 bp DNA sequence downstream (3 0 end) of PAM as a targeting sequence (Figure 1). In the case of BUF1, we designed two spacer/targeting sequences. Note: For LbCas12a guide designing, the targeting sequences must be on the same strand of PAM ('5-TTTV-3'). V indicates A, C or G, but not T. Previous research reported that targeting sequences with GC-content ($40%-60%) provides the highest editing activity for Cas12a (Kim et al., 2017). We advise avoiding targeting sequences with GC content <30% or >70% (Kim et al., 2017).   Note: Do not include PAM sequence in your target reverse oligo.
Note: Various online tools have been developed for genome-wide CRISPR target design. Many are dependent on the organism of interest and must be checked for suitability.
Optional: Commercial guide RNA synthesis kits, compatible with different Cas proteins, are available as an alternative option for preparing gRNA. For example, the NEB EnGenâ sgRNA Synthesis Kit for the S. pyogenes SpCas9 protein can be used for making the corresponding gRNA (NEB, Cat#E3322V).
CRITICAL: If high-quality genome assembly of your isolate is not available, you can check the potential off-target effects of your guides with the commonly used 70-15 genome assembly (Dean et al., 2005).
In vitro guide RNA synthesis and RNP assembly  CRITICAL: Make sure to use the correct concentration of oligos in the reaction. Products from this step do not require purification. The quality of the amplification reaction can be checked by running 2 mL of PCR product on a 2% agarose gel and visualization. The product should be 66 bp.
Optional: Ordering the top and bottom oligos with full sequences (i.e., T7 promoter, LbCas12a mature direct repeat and 23 bp targeting sequences), and annealing them as the DNA template would serve as an alternative option to avoid this PCR step.
6. Guide RNA in vitro transcription. Set up the transcription as below by using HiScribeä T7 High Yield RNA Synthesis Kit (NEB, Cat#E2040S).
Note: Based on our experience, the yield should be at least 50 mg RNA from this step. c. Quantify the RNA quality by NanoDrop spectrophotometer. A 260/280 ratio of 2.0-2.2 following guide RNA transcription and purification indicates successful synthesis. d. Use guide RNA immediately for RNP assembly or store at À80 C for up to 2 months. 8. Assemble the RNP for one reaction in a 0.2 mL PCR tube as indicated below.
Note: Add the LbCas12a protein last to avoid protein precipitation (Fernandez et al., 2018). Assemble the RNP immediately before fungal protoplast transformation.

Donor DNA preparation
Timing: 5-6 h This protocol uses donor DNA for selection, which is integrated at the Cas12a DNA double-strand break site. This step is used to amplify and purify the donor DNA prior to transformation. 9. Amplify the no-homology HYG DNA donor as indicated below Note: Expected PCR amplification size is 1,466 bp. Each fungal protoplast transformation requires $3 mg donor DNA in total. Adjust the number of above PCR reactions based on the total number of transformations you will run. We amplified the HYG DNA donor by using pFGL821 plasmid as DNA template. Other selectable markers (e.g., G418 and Bialaphos) could be used. We amplified G418 coding sequence from plasmid pFGL921 and Bialaphos coding DNA from plasmid pBV9/pSM324 (Kankanala et al., 2007;Zhang et al., 2021). If the gene being edited can be directly used for selection, such as editing FKBP12  or URA3/URA5 as has been shown in Fusarium oxysporum for instance (Wang et al., 2018), the inclusion of donor DNA in the experiment is not necessary.
Note: Selection based on URA3/URA5 has not been established in M. oryzae, and preliminary experiments might be needed to confirm selection based on URA3/URA5 in M. oryzae.
10. Check the amplification quality by running 2 mL of PCR product on a 1% agarose gel. Successful amplification should yield a single product of the expected size. 11. Purify the PCR reaction through Wizard SV Gel and PCR Clean-Up system (Promega). The detailed purification step follows manufacturer's protocols (https://www.promega.com/ -/media/files/resources/protocols/technical-bulletins/101/ wizard-sv-gel-and-pcr-clean-up-system-protocol.pdf?la=en). If there are more products from the amplification product as indicated by step 10, gel purification can be used to isolate the product of interest by using the same Wizard SV Gel and PCR Clean-Up system (Promega). 12. Quantify the DNA quality by NanoDrop spectrophotometer. Donor concentration near 200 ng/ mL-350 ng/mL is preferred.
Note: Store the DNA donor in À20 C freezer until future use.

M. oryzae protoplast preparation
Timing: 5-6 h This step is required to generate the necessary M. oryzae cells to be used for transformation. The protoplasts are obtained by degrading the fungal cell wall, and allows more efficient RNP and nucleic acid transfer.
13. Prepare the protoplast lysing solution freshly as described in ''prepare medium and stock solution'' section. 14. Collect the mycelia from sub-cultured liquid CM through 2-layers of 6 1/2 in Disks Non Gauze Milk Filter papers and funnel. Wash the collected mycelia with a 5 mL 13STC solution. 15. Dry the collected mycelia with extra filter paper, transfer the dry mycelia to 100 mL flask with 25-30 mL protoplast lysing solution. Shake dry mycelia containing the flask at 30 C, 70-80 rpm, for 2.5-3 h under dark to digest the fungal cell wall and release protoplasts.
Note: When transferring the mycelium, manually separate the mass so it is not a single large mass. This will increase the accessibility to the lysing solution.
16. Check the digestion progress and protoplast quality using a hemocytometer under a dissecting microscope. Successful digestion should release many healthy protoplasts, while if many undigested mycelia are observed, extend the digestion time. However, excessive digestion can also cause protoplast collapse (Figure 3). 17. Collect the protoplast by filtering the above lysing reaction containing the fungal protoplasts, through 2-layer miracloth into pre-chilled, 50 mL falcon tube. Wash the lysing debris left in the miracloth with 13STC again to collect all the protoplasts. 18. Centrifuge the 50mL falcon tube with protoplast at 4 C, 5,000 rpm (i.e., 3,829 g in Beckman Coulter-Allegra 25R centrifuge) for 10 min. Note: A visible pellet containing protoplasts should be seen.
19. Discard the supernatant into a biohazard trash container. Gently resuspend the protoplast using 5-10 mL 13STC by pipetting or flicking. 20. Repeat step 18. 21. Discard the supernatant into a biohazard trash container, and gently resuspend the protoplast in 1mL 13STC. Quantify the protoplast concentration using a hemocytometer. Add additional 13STC to adjust the final concentration of protoplasts to 8 3 10 6 to 5 3 10 7 protoplasts/mL. Aliquot 200-250 mL protoplast solution to individual 50 mL falcon tubes for further transformation.
Optional: The protoplasts can be frozen and stored by adding 7% v/v DMSO into the protoplast solution and freezing at À80 C. We recommend performing the protoplast transformation immediately after preparation for best results, but the frozen cultures can still be used for successful editing.
Note: This protocol is strain independent, and we have used it to make protoplasts for various isolates including O-137 and Guy11 (Bao et al., 2017).

M. oryzae protoplast transformation
Timing: 2 days This step transfers the assembled RNP and donor DNA into the fungal cell to allow editing and donor DNA integration.
22. Slowly add 20 mL assembled RNP and 3 mg of no-homology HYG DNA donor into the 50 mL falcon tube containing the aliquoted protoplast. Mix the solution by flicking gently. Place the tube at room temperature for 20-25 min.
Note: If your gene of interest can be used as selection directly, DNA donor is not required in step 22 For example, we have generated fkbp12 (FK506 receptor) mutants by using the selection of drug FK506 without DNA donor successfully .
23. Slowly add 1,000 mL PTC solution to the 50 mL falcon tube. Mix the solution by flicking gently. Place the tube at room temperature for 20-25 min. 24. Slowly add 5 mL liquid TB3 medium into the 50 mL falcon tube. Mix the solution by flicking gently. Cover the 50 mL falcon tube lid with parafilm. 25. Shake 50 mL falcon tube at 28 C, 90 rpm overnight (12-20 h) in the dark. 26. Next day, unseal the 50 mL falcon tube and add melting TB3 medium (near 40 mL for one transformation, 50 C-60 C) with final concentration of 100 mg/mL hygromycin solution. Mix gently, then pour the mixture (overnight protoplast culture + medium) into 150 3 15 mm petri-dish. Wait for about 20 min until complete solidification.
Note: Protoplasts are fragile and require gentle handling. Other antibiotics corresponding to a different no-homology DNA donor can also be used. For example, we have also used G418 at a final concentration: 300 mg/mL. Bialaphos also can be used at a final concentration: 25 mg/ mL in DCM instead of TB3 medium.
27. After the first selection layer has completely solidified, pour a second layer of melting TB3 medium (near 50 mL) with final concentration of 200 mg/mL hygromycin solution into the 150 3 15 mm petri-dish above the first layer. Wait for about 20 min until complete solidification.
Note: For G418 selection, the final concentration for the second layer is 600 mg/mL. Bialaphos also can be used at a final concentration: 100 mg/mL in DCM instead of TB3 medium.
28. Seal the 150 3 15 mm petri-dish with parafilm and incubate the plate upside down at 28 C for 5-7 days under dark.
Note: Transformants using M. oryzae isolate O-137 can typically be seen and transferred at 5 days, while those from isolate Guy11 typically take 7 days. Other isolates will have to be empirically determined.
29. Pick the transformants growing in the top layer for further phenotyping and genotyping. This can be done using a sterile toothpick to move a piece of mycelium to a new plate containing the selection. 30. For BUF1 deletion identification, we cultured the transformants to OTA to check the mycelial color change. For other genes of interest, transformants can be moved to CM with corresponding appropriate selection: 200 mg/mL hygromycin or 500 mg/mL G418. If you use bialaphos selection (final concentration: 100 mg/mL), select DCM instead of CM for screening.

Quick fungal DNA extraction for genotyping
Timing: 5-6 h DNA is extracted from transformants in order to genotype putative DNA edits.
31. Prepare the screw cap tubes with 1.0 mm beads, 400 mL 1M KCL and 400 mL chloroform (added in hood). 32. Add fungal plug (near 1 cm diameter) into the above prepared tubes. (Performed in the biosafety cabinet hood) 33. Put the above tubes in a homogenizer with power 4 3 6 cycles (30 s each cycle) to disrupt the fungal material. 34. Centrifuge 13,200 rpm (i.e.,16,100 g in Eppendorf 5415D centrifuge) 3 10 min 35. Transfer 200 mL supernatant into new labeled tubes 36. Add 120 mL isopropanol in the tube, mix well to precipitate the DNA for at least 10 min (added in hood). 37. Centrifuge 13,200 rpm (i.e.,16,100 g in Eppendorf 5415D centrifuge) 3 10 min. 38. Discard the supernatant and wash the pellet with 500 mL 75% ethanol. 39. Centrifuge 13,200 rpm (i.e.,16,100 g in Eppendorf 5415D centrifuge) 3 10 min. 40. Discard the ethanol and air dry for 10 min. 41. Add 50 mL sterilized water to dissolve the DNA pellet. Note: We prefer to use 1 mL of the dissolved DNA as template for PCR amplification. The DNA extracted by this quick extraction protocol is sufficient for PCR genotyping but should not be considered adequate for other uses such as next-generation sequencing or southern blot.

PCR genotyping
Using the extracted DNA, PCR is used to determine if the gene of interest has been mutated by amplifying multiple regions at the target locus. This allows the researcher to understand and identify a range of possible DNA mutations, including INDELS, donor DNA insertions, and large deletions.
42. We prefer to design three pairs of primer for genotyping; one primer pair amplifies within the gene of interest ($1-2 kb for PCR amplification, the RNP targeting sequence should be included in this amplifying region), and the other two primer pairs amplifying the 5 0 and 3 0 regions of the targeted sequence ($ 0.5 kb for PCR amplification, the reverse primer for 5 0 upstream amplification and the forward primer for 3 0 downstream amplification should locate closely (1-200 bp) to the start codon and stop codon of the gene of interest) (Figure 4). 43. Perform genotyping PCR followed the below program for the gene of interest. We used the BUF1 gene as an example.

EXPECTED OUTCOMES
Gene disruption mutants can be generated by our method with an editing efficiency from $50% to 100% at BUF1 locus ( Figure 5A). Genotyping results suggested that large-scale insertion and deletions are common in the buf1 mutants ( Figure 5B). Please see more detailed analysis and discussion in the original manuscript .

LIMITATIONS
Due to locus-dependent and physiological variables (e.g., gRNA efficiency, chromatin feature, subtelomere/telomere location, and cell cycle), gene-editing efficiency can vary between loci and experiments. (Ceccaldi et al., 2016;Gisler et al., 2019;Huang et al., 2021;Schep et al., 2021;Xue and Greene, 2021). The use of two separate RNPs, targeting DNA regions within proximity to the desired location of editing may increase editing efficiency if single RNP transformation does not generate the desired mutation.

TROUBLESHOOTING
Problem 1 Cannot get the DNA template for T7 in vitro transcription from step 5.

Potential solution
Double check the oligo concentration you used in your system, it requires 100 mM for oligos, which is 103 higher than the normal PCR reaction. Confirm the complementary sequences shared between universal forward primer and target specific reverse primer.
Problem 2 gRNA quality and concentration are low from step 6-7.

Potential solution
A low yield of gRNA could result from the following:

Inefficient gRNA synthesis
Run DNA template before T7 in vitro transcription in 2% agarose gel to confirm the integrity of the initial DNA template. A single, strong signal near 66 bp is expected.
Exactly follow the T7 in vitro transcription step described in this protocol (step 6). Mix all the reaction components well by pipetting before starting the reaction. Extend the reaction time to 18 h for increased gRNA yield.

Inefficient gRNA purification
Follow the purification step described in Monarchâ RNA Cleanup Kit (NEB Cat#T2050L), store the gRNA correctly prior to usage. For this, if we cannot immediately use the gRNA following purification, we store at À80 C for storage.

Problem 3
Poor quality of protoplast from step 21.

Potential solution
Avoid using the melanized mycelia for lysing enzyme digestion.
Prepare the lysing enzyme freshly.
Make sure you use miracloth rather than other filter materials in the filtering step after protoplasts are released.
Gently mix the protoplasts before hemocytometer quantification.
Keep released protoplast on the ice until you start the transformation.

Problem 4
Buff phenotype is not visibly obvious from step 30.

Potential solution
The buff phenotype is most visibly pronounced when M. oryzae is grown on OTA or RPA media. We found that plating the buff strains on CM produces less clear visible color differences when compared to the wild-type strain.

Problem 5
Low gene editing efficiency from step 30.

Potential solution
Many factors contribute to genome editing efficiency.
Fungal protoplast, use freshly prepared protoplast for transformation. We found that freshly prepared protoplast with correct concentration always showed better transformation efficiency than frozen stored protoplast.
Temperature sensitivity, it has been shown before that LbCas12a is a temperature sensitive enzyme, with best editing efficiency at $28 or higher C (Malzahn et al., 2019). Therefore, it is necessary to maintain your lab temperature around 28 C when you perform Cas12a transformation.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, David E. Cook, decook@ksu.edu.

Materials availability
M. oryzae wild-type strains and derived mutants within this study are available upon reasonable request and may require permit.

Data and code availability
This study did not generate any unique datasets or code.