Table 1.
Overview of published reports on CRISPR/Cas-mediated GE of apple.
| CRISPR-tool | Target organism(s) | Target gene(s) | Strategy | Construct design | CRISPR/Cas delivery | Detection of GE events | Resulting edited tissue | GE events | References |
|---|---|---|---|---|---|---|---|---|---|
| SpCas9; LbCas12a | Malus domestica | MdPDS (different cultivars) | Pre-selection of efficient gRNAs by in vitro cleavage assays | Pre-assembled CRISPR RNP complexes | - | - | - | - | Schröpfer et al., 2021 |
| SpCas9 | Malus sieversii (wild apple) | MsPDS | Gene knock-out using two sgRNAs | pYL-CRISPR/Cas9: PUBI-Cas9, AtU3d-P-sgRNA, HygR; pTG-CRISPR/Cas9: P35S-Cas9, AtU6-26-sgRNA(2x), KanR | Stable T-DNA transformation (Agrobacterium tumefaciens) | Albino phenotype (MsPDS knock-out); Sanger sequencing of cloned products | In vitro calli; one chimeric mutated bud | Small deletions (1–28 bp); small insertions (1–2 bp) | Zhang et al., 2021b |
| LbCas12a | M. domestica (“Gala”) | MdPDS | Multiplex targeted mutations with two gRNAs; tracing GE events in chimeric regenerates | AtUbq10-P-LbCas12a,AtU6-P-sgRNAs (2x); KanR | Stable T-DNA transformation (A. tumefaciens) | Albino phenotype (MdPDS knock-out); fluorescent PCR capillary gel electrophoresis; amplicon deep sequencing; Sanger sequencing of cloned products | In vitro shoots; chimeric and homohistont; heterozygous and biallelic mutations; genotypically uniform mutant with biallelic mutations at two target loci | Small deletions (Ø12-13 bp); no large deletions between neighbored target sites; | Schröpfer and Flachowsky, 2021 |
| SpCas9 | M. domestica (and pear) | MdPDS | Dechimerization by adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) | pDE-Cas9: PcUbi4-2-P-SpCas9, MdU3-sgRNA1, MdU6-sgRNA2, KanR | Stable T-DNA transformation (A. tumefaciens) | Albino phenotype (MdPDS knock-out); Sanger sequencing of cloned products | In vitro shoots; complex pattern of editing in T0 shoots; elimination of wild allele in regenerated tissue; chimeric and biallelic | Deletions, substitutions, insertions | Malabarba et al., 2020 |
| SpnCas9-cytidine base editor (CBE) | M. domestica (and pear) | MdALS & MdPDS (“Gala”) | Multiplex base editing of two different reporter genes (MdALS and MdPDS) by two guide RNAs | PcUbi4-3-P-SpnCas9-PmCDA1-UGI, MdU3-sgRNA1, MdU6-sgRNA2, KanR | Stable T-DNA transformation (A. tumefaciens) | Albino phenotype (MdPDS knock-out); growth on chlorsulfuron selection medium (MdALS knock-out); Sanger sequencing of cloned products | In vitro shoots | C-to-T DNA substitutions; other substitutions, deletions | Malabarba et al., 2020 |
| SpCas9 | M. domestica (and grape vine) | MdDIPM4, MdDIPM4 & MdDIPM1 | (1) on-target GE and (2) T-DNA excision from genome by heat-shock inducible Flp/FRT system | AtUbq10-P-SpCas9, AtU6-P-sgRNAs (1-2x),Hsp17.5-E-P-Flp,KanR; flanked by FRT-sites | Stable T-DNA transformation (A. tumefaciens) | Amplicon deep sequencing | Regenerated tissue | (1) Deletion (1–7 bp) in target genes and (2) not reported | Dalla Costa et al., 2020 |
| Cas9 | M. domestica | MdCNGC2 | Knock-out of gene function to improve resistance to Botryosphaeria dothidea; use of two gRNAs | 35S-P-Cas9, MdU6-P-sgRNA1, UBQ10-P-sgRNA2, KanR | Stable T-DNA transformation (A. tumefaciens) | Sanger sequencing of cloned products | In vitro callus | Small deletions and insertions at only one target locus; chimerism and status of mutations not reported | Zhou et al., 2020 |
| SpCas9 | M. domestica (“Gala,” “Golden Delicious”) | MdDIPM4 | (1) Knock-out of gene function to reduce susceptibility to Erwinia amylovora and (2) T-DNA excision from genome by heat-shock inducible Flp/FRT system | AtUbq10-P-SpCas9, AtU6-P-sgRNA,Hsp17.5-E-P-Flp,KanR; flanked by FRT-sites | Stable T-DNA transformation (A. tumefaciens) | Amplicon deep sequencing | Mutated apple plant lines | (1) Mostly small deletions (1–27 bp), small insertions (1 bp); substitution and (2) T-DNA excision reported | Pompili et al., 2020 |
| SpCas9 | M. domestica (“Gala”; and pear) | MdPDS, MdTFL1.1 | Multiplex GE with two gRNAs per target gene; generation of T-DNA-free edited lines | PcUbi4-2-P-SpCas9, MdU3-sgRNA1, MdU6-sgRNA2, KanR | Stable and transient T-DNA transformation (A. tumefaciens) | Albino phenotype (MdPDS knock-out); early flowering (TFL1.1 knock-out); Sanger sequencing of cloned products | In vitro shoots, chimeric tissue, complex editing profiles, biallelic mutations; T-DNA-free edited in vitro shoots | Deletions (1–29 bp), insertions (+1 bp), substitutions, inversion, duplication | Charrier et al., 2019a |
| SpCas9 | M. domestica (and grape vine) | Not specified | Proposal of protocols for: (1) plasmid-mediated GE and (2) DNA-free GE in protoplasts | SpCas9-GFP; KanR | (1) Stable T-DNA transformation (A. tumefaciens) and (2) direct delivery of CRISPR–Cas9 RNPs in protoplasts | PCR-RFLP, Cel-1 assay, heteroduplex mobility analysis (HMA) | - | - | Osakabe et al., 2018 |
| SpCas9 | M. domestica (and grape vine) | MdDIPM1, MdDIPM2, and MdDIPM4 | (1) Determination of in vitro cleavage efficiency of several gRNAs and (2) DNA-free GE in protoplasts | Pre-assembled CRISPR RNP complexes | Direct delivery of CRISPR RNPs in protoplasts | Amplicon deep sequencing | Protoplasts | Deletions, insertions | Malnoy et al., 2016 |
| SpCas9 | M. domestica | MdPDS | Knock-out of gene function using single gRNAs | 35S-P-SpCas9-GFBSD2, AtU6-1-P-sgRNA | Stable T-DNA transformation (A. tumefaciens) | Albino phenotype (MdPDS knock-out); Sanger sequencing (direct or cloned PCR products) | In vitro shoots, chimeric, biallelic | Deletions (1–8 bp), insertions (1 bp) | Nishitani et al., 2016 |