Table 1.
Methods used for screening wheat lines produced using RNA interference or gene editing using CRISPR/Cas9.
| Technology type | Aim | Applied by | |||||||
|---|---|---|---|---|---|---|---|---|---|
| (52) | (25) | (53) | (57) | (21) | (22) | (54) | (58) | ||
| RNAi: | * | * | * | * | * | * | |||
| CRISPR/Cas9: | * | * | |||||||
| Target | A high-transcript-level 169 bp sequence targeting most γ-gliadins; testing RNAi technology | Three plasmid sequences (all with 361 bp chimeric conserved fragments) and their combinations to targeting all α-, γ-, and ω-gliadins | A 313 bp conserved α-gliadin fragment targeting all α-gliadins | Seven plasmid sequence combinations targeting all α-, γ-, and ω-gliadins and LMW glutenin | Two sgRNAs targeting immuno-dominant 33-mer α-gliadin | Six sgRNAs targeting α- and γ-gliadin signal peptide and/or two epitope regions | A 141 bp ω-gliadin fragment targeting ω-1,2 gliadin | A 217 bp fragment with three targets from D-genome α-gliadin genes, targeting α-gliadins | |
| Transformation | Particle bombardment of scutellum tissue of two bread wheat lines; D-Hordein promoter for hpRNAs expression; bar for PPT selection | Gold particle bombardment of immature scutellum tissue of two bread wheat lines; endosperm-specific promoter; bar for PPT-selection | Particle bombardment of immature embryos from a single cultivar; CaMV 35S promoter; nptII gene for kanamycin resistance | Gold particle bombardment of immature scutellum tissue of one bread wheat cultivar; D-Hordein promoter for hpRNAs expression; bar for PPT-selection | Gold particle transformation of scutellum tissue of 2 bread wheat lines and 1 durum wheat cultivar; Ubiquitin 1 promoter from maize; bar for PPT-selection | Agrobacterium transformation of one bread wheat cultivar; ACTIN promoter; nptII for G418-selection | Gold particle bombardment of young embryo-derived callus from a single bread wheat cultivar; HMW-GS Dy10 promoter; bar for PPT-selection | Gold particle bombardment of young embryo-derived callus from Butte 86; maize Ubi1 promoter; bar for PPT-selection | |
| DNA | |||||||||
| PCR | Confirmation of presence of transgene in T0 plants | + | + | + (RT-PCR for RNAi construct expression testing) | + | + | + (presence of Cas9 and all sgRNAs; TR-PCR for expression of Cas9) | + | + |
| Illumina HTP DNA sequencing | Indel characterization and quantification | NA | NA | NA | NA | + | + (applied after gene-enrichment in GlutEnSeq (below) | NA | NA |
| Sanger sequencing | Off-target mutation | NA | NA | NA | NA | + (but not detected) | – | NA | NA |
| ddPCR | HTP gene copy number (variation) assessment | NA | NA | NA | NA | – | + (duplex ddPCR includes reference comparison) | NA | NA |
| GlutEnSeq | Gene enrichment; Identification of large- and medium-sized mutations/deletions | NA | NA | NA | NA | _ | + | NA | NA |
| PROTEIN | |||||||||
| Acid-PAGE; SDS-PAGE | Gluten profile analysis | + (Acid-PAGE) | + (Acid-PAGE) | + (SDS-PAGE) | + (Acid-PAGE of T1 seeds for homozygosity testing; Acid-PAGE and SDS-PAGE of T3) | + | + (Acid-PAGE) | + (SDS-PAGE) | + (SDS-PAGE) |
| 2-DE (2D gel-electrophoresis) | Intensity measurement of specific gluten proteins (in T1 compared with original plant) | – | – | + | – | – | – | + | + |
| MALDI-TOF | Confirmation of PAGE gluten profiles | + (MALDI-TOF MS) | – | + (MALDI-TOF MS for analysis of individual spots from 2-DE) | – | + | – | – | – |
| HPLC | Quantification and characterization of individual gliadin and glutenin protein groups | – | + (RP-HPLC) | + (RP-HPLC) | + (RP-HPLC) | + (RP-HPLC) | – | – | – |
| nanoLC-MSMS | Identification of peptide fragment spectra to be matched to protein sequences in database, and comparison to control | – | – | – | + (LC-MS/MS) | – | + (measuring protein reduction and compensatory effects) | + (MS/MS of isolated 2-DE spots from control plant (to confirm absence of target protein in transformed lines) | + (MS/MS of isolated 2-DE spots from control plant (to confirm absence of target protein in transformed lines) |
| BREAD QUALITY | |||||||||
| SDS sedimentation | Measuring gluten strength for prediction of processing and end-product qualities | – | + | – | + | + | – | + | + |
| Mixing properties | Assessment of dough resistance, development and stability | – | – | – | – | – | – | + (Mixograph) | + (Mixograph) |
| Rheology testing | Maximum resistance of dough to extension (RE) and extensibility (EX) | – | – | + | – | – | – | – | – |
| IMMUNE RESPONSE | |||||||||
| Monoclonal antibodies | Total gluten content in food; 33-mer is target peptide for G12 | + (R5 for total gliadin content) | + (R5 for total gliadin content) | – | G12 (total gluten content) | + R5 and G12 (gluten content and impact on 33-mer) | – | – | – |
| Serum reactivity of CD patients | Immunogenic potential of transgenic lines | – | – | – | – | – | – | + (IgG and IgA antibody reactivity) | + (IgG and IgA antibody reactivity) |
| T cell proliferation response | Testing epitope-specific reactivity | – | + (TG2-treated protease-digested total gluten extract) | – | – | – | – | – | – |
| Food challenge | Coeliac food safety assessment | – | – | – | – | – | – | – | – |
| OTHER ASPECTS RELATED TO MUTANT/TRANSFORMED PLANT PERFORMANCE | |||||||||
| Mutation/transgene stability in T generations | + (measured in T3) | + (aiming at homozygosity) | + (stable integration and expression in T2) | + | + | – | – | – | |
| Absence of transgenes in T generations | NA | NA | NA (3–11 transgene copy numbers in transgenic plant lines) | NA | + | – | NA | NA | |
| Changes in expression of other gene families | – | – | + (RP-HPLC analysis of gluten protein profile: no changes in γ- and ω-gliadin profiles) | – | + (analysis of γ- and ω-gliadin, LMW and whole bread wheat genome by Sanger sequencing) | – | – | + (silencing of some HMW glutenins) | |
| Chromosome number | – | – | – | – | + | – | – | – | |
| Performance (growth; fertility; seed quality/quantity) | + (full fertility; normal grain morphology and weight) | + | + (full fertility; normal seed set and grain morphology; | + (fertility; days to anthesis normal; kernel weight differences) | + | – | + (fertility; normal kernel weight) | + (fertility; normal kernel weight) | |
NA, not applicable.