Figures (3)  Tables (1)

    • Figure 1.  Schematic diagram of genome editing induced by CRISPR/Cas9 system. Cas9 protein cleaves the target sequence (the cut sites are indicated by red triangles) that is complementary with the single guide RNA (sgRNA) and produces double strand breaks (DSB). The DSB could be repaired through non-homologous end joining (NHEJ) or the homology-directed repair (HDR) pathway, resulting in indel (insertion or deletion) or gene targeting mutation.

    • Figure 2.  Schematic overview of base editing and prime editing. In the cytosine base editor (CBE) system, a Cas9 nickase (Cas9n) is fused with a cytidine deaminase and a uracil DNA glycosylase inhibitor (UGI). The CBE could mediate C-to-T substitutions. In the adenine base editor (ABE) system, a Cas9n protein is fused with an engineered Escherichia coli adenosine deaminase (ecTadA), which catalyzes the conversion of adenine (A) to inosine (I). The I is recognized as guanine (G) by DNA polymerase during replication, resulting in A-to-G substitutions. Prime editing (PE) is accomplished by M-MLV-Cas9n-pegRNA complex, in which pegRNA functions as a guide RNA and also provides primer binding site (PBS) and reverse transcriptase (RT) template. The nicked target DNA sequence hybridizes to the PBS, priming reverse transcription of the template into the DNA sequence. The desired mutations within the pegRNA are indicated in purple. PAM, protospacer adjacent motif.

    • Figure 3.  Pipeline of CRISPR/Cas9-mediated genome editing in grape. Both embryogenic callus and protoplasts could be used as materials for grape genome editing. CRISPR/Cas9 reagents such as plasmid constructs and CRISPR/Cas9 ribonucleoproteins (RNPs) can be delivered into the embryogenic callus and protoplasts, respectively. Edited grapevine plants could regenerate from embryogenic callus. Microcalli could be induced from grape protoplasts, but plant regeneration from the protoplasts-induced calli (indicated by broken lines) has not been achieved.

    • Target
      genes      		sgRNA/Cas9 promoterExplantDelivery methodModificationTarget traitsEditing efficiencyOff-target effectReference
      IdnDHAtU6/35SEmbryogenic cells of 'Chardonnay'Agrobacterium-mediated transformationKOTartaric acid synthesis100%NoRen et al.[12]
      MLO-7Not mentionedEmbryogenic calli of 'Chardonnay'PEG-mediated transformationKOPowdery mildew resistance0.1%NDMalnoy
      et al.[13]
      VvPDSAtU6/
      PcUbi4-2      		Embryonic calli of
      'Neo Muscat'      		Agrobacterium-mediated transformationKOAlbino phenotype2%−86%NoNakajima et al.[81]
      VvPDSAtU6/2×35SEmbryogenic cells of 'Chardonnay' and
      '41B'      		Agrobacterium-mediated transformationKOAlbino phenotype22.2%−59.9% ('Chardonnay')
      30.3%−86.6% ('41B')      		NDRen et al.[89]
      VvWRKY52AtU3, AtU6/2×35SProembryonal masses (PEM) of 'Thompson Seedless'Agrobacterium-mediated transformationKOBotrytis cinerea resistance31%NoWang
      et al.[83]
      CCD8AtU6/35SEmbryogenic cells
      of '41B'      		Agrobacterium-mediated transformationKOStrigolactones biosynthesis and shoot branching66.7%NoRen et al.[82]
      VvPR4bAtU6/35SPEM of 'Thompson Seedless'Agrobacterium-mediated transformationKOPowdery mildew resistance68.8%NoLi et al.[85]
      VvMLO3, VvMLO4AtU3, AtU6/2×35SPEM and somatic embryos of 'Thompson Seedless'Agrobacterium-mediated transformationKOPowdery mildew resistance12.8%−38.5%NDWan
      et al.[84]
      TAS4,
      MYBA7      		MtU6.6/
      ZmUbi      		Embryogenic callus
      of rootstock '101-14'      		Agrobacterium-mediated transformationKOAnthocyanin accumulation related to Pierce disease (PD) and Grapevine Red Blotch Virus (GRBV)Not mentionedYesSunitha & Rock[80]
      PDS
      TMT1,
      TMT2      		AtU6, VvU3, VvU6/35S, VvUbiEmbryogenic cells of '41B'Agrobacterium-mediated transformationKOAlbino phenotype
      Sugar accumulation      		23.5%−43.2% (PDS)
      10.4%−20.9% (TMTs)      		NDRen et al.[90]
      Abbreviations: AtU6, Arabidopsis thaliana U6 promoter; MtU6.6, Medicago truncatula U6.6 promoter; VvU6, Vitis vinifera U6 promoter; PcUbi4-2, Petroselinum crispum ubiquitin4-2 promoter; ZmUbi, Zea mays ubiquitin promoter; VvUbi, V. vinifera ubiquitin promoter; 35S, CaMV 35S promoter; KO, Knock out; ND, Not determined.

      Table 1.  Applications of the CRISPR/Cas9 system in grape.