Protocol for Genome Editing to Produce Multiple Mutants in Wheat

Summary Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat (Triticum aestivum) cv. Fielder. Using Agrobacterium-delivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated. The technique for acceleration of generation advancement with embryo culture reduces time for mutant production. The mutants produced by this protocol can be used for the analysis of gene function and crop improvement. For complete details on the use and execution of this protocol, please refer to Abe et al. (2019).


SUMMARY
Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat (Triticum aestivum) cv. Fielder. Using Agrobacteriumdelivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated. The technique for acceleration of generation advancement with embryo culture reduces time for mutant production. The mutants produced by this protocol can be used for the analysis of gene function and crop improvement. For complete details on the use and execution of this protocol, please refer to Abe et al. (2019).

BEFORE YOU BEGIN
Facilities: Transformation of wheat requires immature embryos as explants; therefore, researchers must first grow wheat plants. Due to the lower efficiency of genome editing in wheat compared with in model species, we recommend using facilities that can continuously grow wheat plants in appropriate conditions throughout the year.

Growing Wheat Plants to Obtain Immature Embryos
Timing: about 15 weeks (depending on the season) 1. Sow 6 seeds of wheat cv. Fielder in 18-cm plastic pots containing a 2:1 mixture of Sakata Supermix A (fine peat moss) and Nippi fertilized granulated soil. In one pot, add 2.6 L of the soil mixture and 2.6 g of the controlled release fertilizer Osmocote Exact Mini 3-4M 15-9-11+2MgO+TE. 2. Grow the plants in a glasshouse ( Figure 1A left) at day/night temperatures of 16 C/10 C under an 8 h light/16 h dark photoperiod. After 10-12 weeks (depending on the season), transfer the plants just before heading to a controlled environmental chamber ( Figure 1A right) with adjusted day/ night temperatures of 20 C/13 C under a 14h light (300-500 mmol m À2 s À1 )/10h dark photoperiod without artificial humidity control.
Note: Here is an example of the conditions we use for growing wheat plants. Growing wheat plants are shown in Figure 1B. To generate a continuous supply of embryos, we recommend sowing new batches of seeds every week. DNA  Data and Code Availability This study did not generate any unique datasets or code.

MATERIALS AND EQUIPMENT
Note: Agarose type I and Hygromycin B are the specific chemicals that we recommend should be used. Alternatives should not be substituted.
Note: To excise the embryo axis from the immature embryos, a Futaba No. 19 is more suitable as a scalpel blade, but a Feather No. 14 can be used as well.

Make Stock Solutions
Timing: 1-2 days Biotin (0.1 mg/mL) Dissolve 1 mg (+)-Biotin in 10 mL of distilled water, and store in 0.1-mL aliquots at À20 C in the dark.
AgNO 3 (100 mM) Dissolve 169.9 mg AgNO 3 in 10 mL of distilled water. Sterilize with a 0.22-mm polyethersulfone (PES) filter, and store in 0.1-mL aliquots at À20 C in the dark.
Picloram (2.2 mg/mL) Add 1 N NaOH dropwise to 110 mg picloram until completely dissolved. Make up to 50 mL with distilled water. Sterilize with a 0.22-mm PES filter, and store in 1-mL aliquots at À20 C. Total 100 mL Store in 1-mL aliquots at À20 C in the dark.

OPEN ACCESS
Ascorbic Acid (100 mg/mL) Dissolve 5 g L(+)-ascorbic acid in 50 mL of distilled water. Sterilize with a 0.22-mm PES filter, and store in 1-mL aliquots at À20 C in the dark.
Carbenicillin (250 mg/mL) Dissolve 5 g carbenicillin disodium salt in 20 mL of distilled water. Sterilize with a 0.22-mm PES filter, and store in 1-mL aliquots at À20 C in the dark.
Cefotaxime (250 mg/mL) Dissolve 5 g cefotaxime sodium salt in 20 mL of distilled water. Sterilize with a 0.22-mm PES filter, and store in 1-mL aliquots at À20 C in the dark.
Zeatin (5 mg/mL) Add 1 N HCl dropwise to 250 mg zeatin until completely dissolved. Make up to 50 mL with distilled water. Sterilize with a 0.22-mm PES filter, and store in 1-mL aliquots at À20 C in the dark.
Indole-3-butyric Acid (IBA, 1 mg/mL) Add 1 N NaOH dropwise to 50 mg indole-3-butyric acid until completely dissolved. Make up to 50 mL with distilled water and store at 4 C.
Ampicillin (50 mg/mL) Dissolve 1 g ampicillin sodium in 20 mL of distilled water. Sterilize with a 0.22-mm PES filter, and store in 1-mL aliquots at À20 C in the dark.
Spectinomycin (15 mg/mL) Dissolve 300 mg spectinomycin dihydrochloride pentahydrate in 20 mL of distilled water. Sterilize with a 0.22-mm PES filter, and store in 1-mL aliquots at À20 C in the dark.
Kanamycin (50 mg/mL) Dissolve 1 g kanamycin sulfate in 20 mL of distilled water. Sterilize with a 0.22-mm PES filter, and store in 1-mL aliquots at À20 C in the dark.

60% (v/v) Glycerol
Measure out 60 mL of glycerol. Make up to 100 mL with distilled water. Autoclave at 121 C for 20 min, and store at 20-25 C.

Make Media
Timing: 1-2 days  Inoculum (WMS-inf) Medium Immediately before use, add 1 mL of 100 mM AS to 1 mL of WMS-liq medium.

Reagent
Weight/Volume Peptone 10 g Yeast extract 10 g NaCl 5 g ddH 2 O Total 1 L Adjust pH to 7.0. Make up the volume to 1 L, sterilize with the Vacuum Filter/Storage Bottle System, and store at 4 C.

Reagent
Weight/Volume

Reagent
Weight AgNO 3 (100 mM) 50 mL Pour 20 mL of medium into a 90-mm petri dish, and air-dry in the laminar flow clean bench for 1 h. Seal with parafilm and store at 20-25 C in the dark. The medium can be stored for up to 3 weeks.

Reagent
Weight/Volume Adjust pH to 5.8 Agarose (type I) 8 g Make up the volume to 1 L, and autoclave at 121 C for 20 min Cool to 50 C, and then add the reagents below AS (100 mM) 2 mL AgNO 3 (100 mM) 50 mL Pour 20 mL of medium into a 90-mm petri dish, and air-dry in the laminar flow clean bench for 1 h. Seal with parafilm and store at 4 C in the dark. The medium can be stored for up to 6 weeks. Hygromycin (50 mg/mL) 0.6 mL Pour 25 mL of medium into a 90-mm petri dish, and air-dry in the laminar flow clean bench for 1 h. Seal with parafilm and store at 20-25 C in the dark. The medium can be stored for up to 3 weeks.

Reagent
Weight/Volume Agarose (type I) 5 g Make up the volume to 1 L, and autoclave at 121 C for 20 min Cool to 50 C, and then add the reagents below 2,4-D (1 mg/mL) 0.5 mL Picloram (2.2 mg/mL) 1 mL Ascorbic acid (100 mg/mL) 1 mL Carbenicillin (250 mg/mL) 1 mL AgNO 3 (100 mM) 50 mL Hygromycin (50 mg/mL) 0.3 mL Pour 25 mL of medium into a 90-mm petri dish, and air-dry in the laminar flow clean bench for 1 h. Seal with parafilm and store at 20-25 C in the dark. The medium can be stored for up to 3 weeks.

Reagent
Weight/Volume To produce genome-edited multiple-recessive mutants in wheat, the target sequences must have a protospacer adjacent motif (PAM) for Cas9 nuclease and a restriction site for verifying the mutations.

Reagent Weight/Volume
Adjust pH to 5.8 Phytagel 3 g Make up the volume to 1 L, and autoclave at 121 C for 20 min Cool to 50 C, and then add the reagents below Zeatin (5 mg/mL) 1 mL Carbenicillin (250 mg/mL) 0.5 mL Hygromycin (50 mg/mL) 0.6 mL Pour 20 mL of medium into a 90-mm petri dish, and air-dry in the laminar flow clean bench for 1 h. Seal with parafilm and store at 20-25 C in the dark. The medium can be stored for up to 3 weeks.

Reagent
Weight/Volume Make up the volume to 1 L, and autoclave at 121 C for 20 min Cool to 50 C, and then add the reagent below Hygromycin (50 mg/mL) 0.3 mL Pour 20 mL of medium into a 90-mm petri dish, and air-dry in the laminar flow clean bench for 1 h. Seal with parafilm and store at 20-25 C in the dark. The medium can be stored for up to 3 weeks. Note: According to Liang et al. (2016), three stem-loop structures, i.e. loops RAR, 2, and 3, are crucial for gRNA stability and activity. Therefore, a gRNA that can create such a structure should be chosen. We also confirmed the importance of the predicted secondary structure of designed gRNA in wheat (Kamiya et al., 2020).

Half-Strength MS Medium
4. Check for potential off-target sites at CRISPR-P 2.0 (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/ CRISPR). Reject gRNAs with potential off-target sites. 5. Conduct in vitro cleavage assays to exclude inferior sgRNAs, using the Guide-it Complete sgRNA Screening System. All steps should be performed according to the manufacturer's instructions. 6. Design primer sets for amplifying the fragments, including the target sites. Websites for primer design are useful, for example Primer3Web (https://primer3plus.com/primer3web/ primer3web_input.htm).
Note: The success of genome editing depends on the proper function of the designed gRNA.
Although the nucleotide sequences that are conserved among homoeologs in cv. Chinese Spring are almost identical to those in cv. Fielder, the target sites of cv. Fielder should be sequenced to confirm this. The whole-genome shotgun data for the null-segregant, cv. Fielder (negative control), and the transgene-positive T 1 plant #1-8 (positive control) have been deposited in the DDBJ Sequence Read Archive under BioProject Accession PRJDB7455 (Abe et al. 2019). The sequence of Fielder can be generated by mapping whole-genome shotgun data of cv. Fielder (negative control) onto the reference target sequence (e.g. cv. Chinese Spring) to be edited. The candidate target site is better to have a restriction enzyme site that overlaps with the fourth nucleotide from the PAM; this makes it easier to detect mutations by PCR-RFLP using the restriction enzyme. If this site is not available, the PCR products, including the target sites of all homoeologs, should be sequenced to detect the mutations.

Timing: 2 weeks
Details of the construction method differ, depending on the vectors used to express Cas9 and the gRNA. We describe an example of the vector construction strategies used in Abe et al. 2019.
Pause Point: The digestion may be incubated for 16-24 h.
9. Run the digested products on a 1% agarose gel in TAE buffer, and purify the digested vector using a QIAquick Gel Extraction Kit. 10. Measure the concentration of the digested vector using a microvolume UV spectrophotometer, and dilute the digested vector to 10 ng/mL. 11. Combine 1 mL of each oligo DNA (100 mM) and 48 mL of sterilized distilled water. Boil for 5 min and leave at 20-25 C for 20 min to anneal the oligonucleotide. 12. Ligate the annealed gRNA oligonucleotide into the digested vector using a Rapid DNA Ligation Kit.
13. Transform 5 mL of the ligation reaction using an E. coli DH5a Competent Cells Kit. Plate the cells onto LB agar containing 50 mg/L ampicillin, and incubate at 37 C for 16 h. 14. Inoculate four colonies into separate 3-mL aliquots of LB medium containing 50 mg/L ampicillin and grow the cells at 37 C with vigorous shaking for 16 h. 15. Extract plasmid DNA with a QIAprep Miniprep kit. 16. Sequence plasmids with the OsU6-2F (5 0 -TGCTGGAATTGCCCTTGGATCATGAACCAA-3 0 ) primer using a BigDye Terminator v3.1 Cycle Sequencing Kit to verify that the clones harbor the designed gRNA.

Cloning a gRNA Expression Cassette into the Binary Vector
17. Digest 2 mg of the binary vector, pZH_gYSA_PubiMMCas9, and the completed gRNA vector using 1 mL each of the restriction enzymes AscI and PacI, and 2 mL of CutSmart buffer in a 20 mL final volume, and incubate at 37 C for 3 h.
Pause Point: The digestion may be incubated for 16-24 h.
18. Run the digested products on a 1% agarose gel in TAE buffer, and purify the digested vector and gRNA expression cassette using a QIAquick Gel Extraction Kit. 19. Quantify the digested vector and gRNA expression cassette concentration using a microvolume UV spectrophotometer. 20. Ligate the digested gRNA insert into the digested binary vector using a Rapid DNA Ligation Kit.

Reagent
Volume ( (Hood et al., 1986) into 10 mL of YEP medium containing 50 mg/L kanamycin and grow the cells at 28 C with vigorous shaking for 30 h. 26. Collect bacterial cells by centrifugation and resuspend in 1 mL of pre-cooled 20 mM CaCl 2 . 27. Freeze aliquots of 100 mL in liquid nitrogen. The rest can be stored at À80 C. 28. Add 0.5-1.0 mg of plasmid DNA to the frozen cells directly, and thaw at 37 C for 5 min. 29. Dilute in 1 mL of YEP medium, and shake at 28 C for 2 h. 30. Plate the cells onto YEP agar containing 75 mg/L spectinomycin, and incubate at 28 C for 2 days. 31. Pick a single spectinomycin-resistant A. tumefaciens colony and culture it in 1 mL of YEP medium containing 75 mg/L spectinomycin at 28 C with vigorous shaking for 20-24 h. 32. Add 1 mL of 60% (v/v) glycerol to the culture, and vortex thoroughly. 33. Store aliquots of 20 mL at À80 C.

Agrobacterium-Mediated Transformation
Timing: 3 months Agrobacterium-mediated transformation is a straightforward approach to plant transformation that results in the insertion of only one or a few copies of the transgene. Producing the transgene-free genome-edited mutant is efficiently accomplished by crossing the resulting transformed plant with a wild-type plant and screening for progeny that contain the mutation but lack the transgene in the F 2 generation.
Infection of Immature Embryos with A. tumefaciens 34. Add 10 mL of A. tumefaciens glycerol stock (step 33) to 10 mL of MG/L medium (no antibiotics), and grow cells at 28 C with vigorous shaking for 20-24 h. 35. Collect bacterial cells by centrifugation and resuspend to a cell density of 0.4 of A 660 in WMS-inf medium. 36. Collect immature seeds at the right developmental stage from panicles about 15 days after anthesis (DAA) (Video S1).

OPEN ACCESS
CRITICAL: The use of immature embryos at the correct developmental stage is a critical factor, and the size of the embryos is a very good indicator of the stage. Immature embryos that are 2.0-2.5 mm in length along the long axis are optimal for transformation. The time (DAA) required for embryos to reach the optimum stage differs, depending on the genotype and the season. Because wounding of immature embryos is detrimental for infection by A. tumefaciens and subsequent tissue culture, handle the immature embryos carefully up to the step for excising the embryo axis (step 50).
37. Remove the glumes, lemma, and palea with forceps (Video S1). 38. Sterilize immature seeds with 70% ethanol for 1 min and 0.5% sodium hypochlorite for 15 min, and then wash three times with sterilized distilled water. 39. Isolate immature embryos from the immature seeds under a stereoscopic microscope and transfer the embryos into 2.0 mL of WMS-liq medium in a 2.0-mL microcentrifuge tube (Video S1). Taking into account subsequent handling of the embryos, it is recommended to place fewer than 100 embryos into one microcentrifuge tube. 40. Invert the microcentrifuge tube several times and remove the medium. 41. Add 1.9 mL of WMS-liq medium. 42. Centrifuge the microcentrifuge tube with a fixed-angle rotor with a maximum radius of 83 mm at 20,0003g at 4 C for 10 min. 43. Remove the medium from the microcentrifuge tube and add 1.0 mL inoculum (step 35) (Video S1). 44. Invert the microcentrifuge tube frequently for 30 s (Video S1). 45. Incubate at 20-25 C for 5 min. 46. Transfer the immature embryos to WMS-AS medium using a 60-mm petri dish. Although the inoculum will be absorbed by the culture medium, any excess inoculum should be removed with forceps (Video S1).
Note: About 50 embryos can be placed onto a single plate. We recommend starting with 10 embryos per plate and increasing the number up to 50 embryos depending on the level of skill. When the plate is left open for a long time to excise the embryo axis (step 50), the embryo will gradually dry out and die.  Table).

Detection and Genotyping of the Mutation
Timing: 3 days 60. Amplify the targeted region of the transformants by PCR with the conserved primer set that recognizes all three homoeologs (TaQsd1-ABD in Key Resources Table). 61. Digest 5 mL of the PCR products using 0.5 mL of PstI enzyme with 1 mL of CutSmart buffer in a 10-mL final volume. Incubate at 37 C for 3 h. 62. Run the products on a 2% agarose gel with Gel Red. 63. Visualize the results on a UV transilluminator. 64. Identify which homoeologs contain a mutation using three specific primer sets (such as TaQsd1-A, TaQsd1-B, and TaQsd1-D in Key Resources Table), one for each homoeolog. 65. Sequence the undigested fragments of three specific primer sets using a BigDye Terminator v3.1 Cycle Sequencing Kit to identify the type of mutation.

Acceleration of Generation Advancement with Embryo Culture
Timing: 2 weeks Isolating immature embryos and germinating them on medium, rather than waiting for seeds to mature, allows the next generation of plants to be obtained more rapidly. This technique can also be applied to immature seeds resulting from crosses.  71. Confirm the segregation of the transgene by PCR with hpt and Cas9 primer sets, and identify which homoeologs contain a mutation using three specific primer sets as described above.
Note: Using this technique, we successfully produced a transgene-free triple-recessive spring wheat mutant in a short time. Only 14 months were required from the infection of immature embryos to obtaining the homozygous F 3 mutant seeds of a cross progeny (Abe et al., 2019).

EXPECTED OUTCOMES
Genome-edited, transgene-free, triple-recessive mutants can be produced in hexaploid wheat. We confirmed that the transgene, which was located in a maximum of five loci, can be segregated away simultaneously by crossing with the wild-type plant in the next (F 1 ) generation (Abe et al., 2019). In our conditions, the transformation efficiency of treated immature embryos ranged between 2-5%, and 7-39% of the transformants, depending on the target sequence, all contained mutations at any of the target sites (Kamiya et al., 2020).

LIMITATIONS
An efficient Agrobacterium-mediated transformation technique (Ishida et al., 2015) is currently available only for cv. Fielder and a limited range of alternative cultivars. Protocols are limited to investigating the roles of genes in those genotypes. Mutations could be transferred from these cultivars to another by standard crossing. Our technique for generation advancement via embryo culture can also be used to accelerate the introduction of multiple mutations into other genetic backgrounds.
The genome sequence of wheat (IWGSC (The International Wheat Genome Sequencing Consortium), 2018) helps to precisely identify T-DNA fragments within the genome. We mapped four T-DNA integrations to their respective chromosomal positions of sub-genomes by sequencing the border junctions of the T-DNA and the wheat genome. This approach was quite difficult before the release of the wheat reference genome, due to the sequence similarity among homoeologs.
The high-quality genome sequence of the target haplotype might be needed as an initial step for genome editing in wheat. We previously sequenced bacterial artificial chromosome clones from cv. Chinese Spring harboring wheat orthologs of barley (Hordeum vulgare) Qsd1 to re-sequence one in cv. Fielder and to design experiments for genome editing.
We also examined whether this Agrobacterium-based genome editing system is generally applicable for other genes. We reproduced our results in wheat cv. Fielder with other genes. However, we had limited success for mutation induction, and obtained mutations only in a few genes with a lower frequency of multiple mutations in a single plant. For example, we attempted to mutate targeted wheat orthologs of barley SD2 (TaQsd2), but were not successful (unpublished data).

TROUBLESHOOTING Problem
The transformation success rate is low.

Potential Solution
The wheat embryos are very sensitive to environmental conditions. If the transformation success rate is low, check the plant growth conditions before investigating other aspects of the protocols, such as vectors and media compositions. We recommend testing the steps for infection of explants with A. tumefaciens using a reporter gene such as b-GLUCURONIDASE (GUS). Monitoring the level of expression of a transgene in immature embryos at the resting culture stage, 7 days after infection with A. tumefaciens, provides useful information for optimizing the transformation protocol.

Problem
Genome-edited plants cannot be obtained.

Potential Solution
The success of genome editing depends on the proper functioning of the designed gRNA. If genome-edited plants cannot be obtained, please redesign the gRNA first. Deep learning-based systems to predict CRISPR/Cas9 sgRNA on-target cleavage efficiency, such as DeepSpCas9