Table 1. Agricultural applications of the use of CRISPR systems in the 52 articles studied (2014–2017).
Plant species | Application perspectives | Targeted sequence(s) | Molecular functions | Delivery method//main strategy | Transgene-free plants studied (yes/no) | Reference |
---|---|---|---|---|---|---|
BIOTIC STRESS TOLERANCE | ||||||
Virus stress tolerance | ||||||
Model plants | ||||||
Arabidopsis thaliana | Potyvirus resistance (TuMV) | eIF(iso)4E, member of the eukaryotic translation initiation factor | Recessive resistance alleles against various potyviruses | Agrobacterium-mediated transformation with a Cas9/gRNA recombinant plasmid binary vector (floral dipping) // gene knockout with Cas9/gRNA | Yes | [9] |
Arabidopsis thaliana and Nicotiana benthamiana | Beet severe curly top virus (BSCTV) tolerance | 43 candidate sites in coding or non-coding sequences of the BSCTV genome for transient expression assays and selection of two sites for transgenic lines induction | Virus replication mechanism | Agrobacterium-mediated transformation of leaves with Cas9/gRNA expression plasmid vectors // gene knockout with Cas9/gRNA | No | [7] |
Nicotiana benthamiana | Tomato yellow leaf curl virus (TYLCV) resistance | Coding and non-coding sequences of TYLCV | Virus replication mechanism | Agrobacterium-mediated transformation of leaves with a TRV RNA replicon for the delivery of gRNAs into Cas9 overexpressing plants // gene knockout with Cas9/gRNA | No | [14] |
Virus tolerance | AGO2 gene | Contribution to antiviral immunity (virus-specific antiviral role of AGO2 gene) | Agrobacterium-mediated transformation of leaves with Cas9/gRNA expression plasmid vectors // gene knockout with Cas9/gRNA | No | [20] | |
Crops | ||||||
Cucumis sativus | Ipomovirus immunity, tolerance to the Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W | eIF4E (eukaryotic translation initiation factors 4E) | Host factors for RNA viruses, recessive resistance alleles against viruses | Agrobacterium-mediated transformation of cut cotyledons (without embryo) with binary vector containing Cas9/gRNA // gene knockout with Cas9/gRNA | Yes | [21] |
Fungus stress tolerance | ||||||
Crops | ||||||
Oriza sativa | Blast (caused by Magnaporthe oryzae) tolerance | Ethylene responsive factor ERF transcription factor gene OsERF922 | Involved in the modulation of multiple stress tolerance | Agrobacterium-mediated transformation of embryogenic calli with Cas9/gRNA-expressing binary vectors // single and multiplex gene knockout with Cas9/gRNA | Yes | [22] |
Solanum lycopersicum | Powdery mildew resistance | SlMlo gene | Major contributor to powdery mildew susceptibility | Agrobacterium-mediated transformation of cotyledons with Cas9/gRNA expression plasmid vectors // gene knockout with Cas9/gRNA | Yes | [23] |
Triticum aestivum | Powdery mildew (Blumeria graminis f. sp. Tritici) resistance | One of the three mildew-resistance locus (MLO) homeologs in bread wheat: TaMLO-A1 allele | Encode a protein that was shown to repress defenses against powdery mildew diseases | Particle bombardment with Cas9/gRNA expressing plasmid into immature wheat embryos // gene knockout with Cas9/gRNA | Yes | [6] |
Bacteria stress tolerance | ||||||
Crops | ||||||
Citrus paradisi | Citrus canker (caused by Xanthomonas citri subspecies citri (Xcc)) tolerance | PthA4 effector binding elements (EBEs) in the Type I CsLOB1 promoter (EBEPthA4-CsLOBP) of the CsLOB1 (Citrus sinensis lateral organ boundaries) gene | CsLOB1: susceptibility gene for citrus canker CsLOB1 gene expression induced by the binding of the pathogenicity factor PthA4 to the EBEPthA4-CsLOBP | Agrobacterium-mediated transformation of epicotyl with Cas9/gRNA expression plasmid vectors // gene knockout with Cas9/gRNA | No | [24] |
Citrus sinensis Osbeck | Canker resistance | CsLOB1 promoter | Susceptibility gene CsLOB1 promoter in citrus | Agrobacterium-mediated epicotyl transformation // gene knockout with Cas9/gRNA | No | [25] |
Oryza sativa | Bacterial blight (caused by Xanthomonas oryzae pv. oyzae) tolerance | Sucrose transporter gene OsSWEET13 | Disease-susceptibility gene for PthXo2 (TAL effector gene of X. oryzae pv. oryzae) | Agrobacterium-mediated transformation of embryogenic callus with Cas9/gRNA expression plasmid vectors // gene knockout with Cas9/gRNA | No | [26] |
ABIOTIC STRESS TOLERANCE | ||||||
Herbicide tolerance | ||||||
Model plants | ||||||
Arabidopsis thaliana | Cold, salt and drought stress tolerance | UDP-glycosyltransferases UGT79B2 and UGT79B3 | UGT family responsible for transferring sugar moieties onto a variety of small molecules and control many metabolic processes; UGT79B2 and UGT79B3 could be induced by various abiotic stresses | Agrobacterium-mediated transformation with a Cas9/gRNA recombinant plasmid binary vector via floral dipping // gene knockout with Cas9/gRNA | No | [27] |
Glufosinate resistance and reduced trichomes formation | BAR gene GL1 gene |
BAR gene confers glufosinate resistance. GL1 gene is required for trichomes formation. |
Agrobacterium-mediated transformation with Cas9/gRNA plasmid vectors (floral dipping) // gene knockout with Cas9/gRNA | Yes | [28] | |
Lotus japonicus | Bioavailability of soil organic nitrogen and capability to accommodate nitrogen-fixing bacteria intracellularly to fix its own nitrogen | Single and multiple symbiotic nitrogen fixation (SNF) genes: simbiosis receptor-like kinase (SYMRK), leghemglobin loci (LjLb1, LjLb2, LjLb3) | Involved in symbiotic nitrogen fixation | A. tumefaciens and A. rhizogenes-mediated transformation containing the appropriate plasmids // gene knockout with Cas9/gRNA | No | [3] |
Crops | ||||||
Linum usitatissimum | Glyphosate tolerance | 5′-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) | EPSPS genes encode a protein in the Shikimate pathway that participates in the biosynthesis of aromatic amino acids; EPSPS is a target for the glyphosate where it acts as a competitive inhibitor of the binding site for phosphoenolpyruvate | Protoplast transfection with ssODN and CRISPR-Cas9 plasmid // gene replacement | No | [15] |
Oryza sativa | Herbicide resistance | C287 gene | The C287Tgene mutation endows rice plants with resistance to the herbicide imazamox (IMZ) | Agrobacterium-mediated transformation // CRISPR-Cas9-mediated multiplex genome editing | No | [29] |
Herbicide tolerance | Acetolactate synthase (ALS) gene | Involved in the ALS biosynthesis (amino acid biosynthesis) | Co-transformation of rice calli through particle bombardment with Cas9/gRNA expression plasmid vector and oligonucleotide donor // gene replacement with a donor template | No | [16] | |
Glyphosate tolerance | 5′-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) | Involved in the biosynthesis of aromatic amino acids | Co-transformation of rice calli through particle bombardment with Cas9/gRNA expression plasmid and donor plasmid // gene insertion and replacement with a donor template | Yes | [30] | |
Solanum tuberosum | Reduced susceptibility to ALS-inhibiting herbicides | Acetolactate synthase 1 (ALS1) | Involved in the acetolactate synthase biosynthesis (amino acid biosynthesis) | Agrobacterium-mediated transformation for GVR-mediated delivery of CRISPR–Cas9 system and donor template // gene knockout and replacement | No | [18] |
Salt stress tolerance | ||||||
Crops | ||||||
Oryza sativa | Salt stress tolerance | GT-1 element in the salt induction of OsRAV2 (key regulatory regions in its promoter) | RAV subfamily involved in developmental processes such as the brassinosteroid response, leaf senescence and flowering time and also in plant responses to abiotic stress including high salinity | Agrobacterium-mediated transformation of leaves with Cas9gRNA plasmid expression vector // gene knockout with Cas9/gRNA | No | [1] |
Drought stress tolerance | ||||||
Crops | ||||||
Zea mays | Improved grain yield under field drought stress conditions | ARGOS8 | Negative regulator of ethylene responses | Co-transformation of immature embryos by particle bombardment with DNA repair template Cas9-sgRNA expression plasmids // gene insertion or replacement with a donor template | No | [17] |
YIELD, BIOFORTIFICATION AND CONSERVATION PARAMETERS | ||||||
Yield | ||||||
Crops | ||||||
Brassica oleracea and Hordeum vulgare | Pod shatter and control of dormancy | HvPM19 BolC.GA4.a |
Positive regulator of grain dormancy Involved in pod valve margin development |
Agrobacterium-mediated transformation // gene knockout with Cas9/gRNA | Yes | [31] |
Dendrobium officinale | Lignocellulose biosynthesis | C3H, C4H, 4CL, CCR and IRX genes | Target genes are involved in the lignocellulose biosynthesis pathway | Agrobacterium-mediated transformation // gene knockout with Cas9/gRNA | No | [32] |
Nicotiana tabacum, sylvestris and tomentosiformis | Regulation of axillary bud growth | NtPIN4 gene | Involved in auxin biosynthesis | Agrobacterium-mediated transformation of leaves // gene knockout with Cas9/gRNA | Yes | [33] |
Oryza sativa | Starch synthesis pathway in rice pollen | Plastific large subunit of ADP-glucose pyrophosphorylase (OsAGPL4) | Involved in the starch synthesis pathway | Agrobacterium-mediated transformation with Cas9/gRNA plasmid expression vector // gene knockout with Cas9/gRNA | No | [34] |
Regulation of pollen tube growth and integrity | Rice member of plant-specific receptor-like kinase CrRLK1LS subfamily, ruptured pollen tube (RUPO) | Receptor-like kinase (RUPO) as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction | Agrobacterium-mediated transformation of embryo-derived rice callus with Cas9/gRNA expression plasmids // gene knockout with Cas9/gRNA | No | [35] | |
Grain yield performance | Grain size3 (GS3) and Grain number 1a (Gn1a) | Grain yield QTLs identified to regulate grain size and grain number | Agrobacterium-mediated transformation with Cas9/gRNA plasmid expression vector // gene knockout with Cas9/gRNA | Yes | [36] | |
Grain weight | Grain width 2 (GW2), grain width 5 (GW5) and thousand-grain weight (TGW6) | Three major genes that negatively regulate rice grain weight | Agrobacterium-mediated transformation with Cas9/gRNAs plasmid expression vector // CRISPR–Cas9-mediated multiplex genome editing | Yes | [37] | |
Development of japonica photo-sensitive genic male sterile rice lines | Carbon starved anther (CSA) | One important locus for regulating photoperiod-controlled male sterility in japonica rice | Agrobacterium-mediated transformation with two plasmids into calli // gene knockout with Cas9/gRNA | Yes | [38] | |
Enhanced grain number, dense erect panicles, larger grain size | Cytokinin dehydrogenase2 (Gn1a), γ-subunit of G protein (DEP1), γ-subunit of G protein (GS3) and squamosa promoter binding protein (IPA1) | Regulators of grain number, panicle architecture, grain size and plant architecture | Agrobacterium-mediated transformation with Cas9/gRNA plasmid expression vectors // gene knockout with Cas9/gRNA | Yes | [39] | |
Maintenance and determinacy of the flower meristem | FLORAL ORGAN NUMBER2 (FON2) gene OsMADS3 gene |
Involved in meristem maintenance and in stamen specification | Agrobacterium-mediated transformation of calli // gene knockout with Cas9/gRNA | No | [40] | |
Rice caryopsis development | OsSWEET11 gene | Sugar transporter | Agrobacterium-mediated transformation of leaves // gene knockout with Cas9/gRNA | No | [41] | |
Stomatal developmental | EPFL9 gene | Positive regulator of stomatal developmental pathway | Agrobacterium-mediated transformation of immature embryos // gene knockout with CRISPR–Cas9/Cpf1 system | Yes | [42] | |
Developing marker-free transgenic plants | GUS gene | Marker gene | Agrobacterium or gene gun with a construct expressing Cas9 and two gRNAs // gene knockout with Cas9/gRNA | No | [43] | |
Rice development | MPK1 and MPK6 gnes | Essential genes for rice development | Agrobacterium-mediated transformation of rice calli // gene knockout with Cas9/gRNA | Yes | [5] | |
Regulation of seed development | MEGs and PEGs genes | Involved in the regulation of nutrient metabolism and endosperm development | Agrobacterium-mediated transformation // gene knockout with Cas9/gRNA | No | [44] | |
Breeding of early-maturing rice cultivars | Hd2, Hd4 and Hd5 genes | Flowering suppressors in Ehd1-dependent photoperiodic flowering pathway and major genes that negatively control the heading date of rice varieties grown in the north of China | Agrobacterium-mediated transformation // gene knockout with Cas9/gRNA | No | [45] | |
Solanum lycopersicum | Generation of parthenocarpic tomato plants | SlIAA9 gene | A key gene controlling parthenocarpy | Agrobacterium-mediated transformation of leaves // gene knockout with Cas9/gRNA | No | [46] |
Taraxacum kok-saghyz | Rubber biosynthesis in hairy roots | TK 1-FFT (fructan:fructan 1-fructosyltransferase) | Implicated in inulin biosynthesis (antagonist of rubber production) | TK plantlets inoculated with Agrobacterium rhizogenes harbouring a plasmid encoding Cas9/gRNA (wounded surface of the plantlets dipping) // gene knockout with Cas9/gRNA | No | [47] |
Zea mays | High-frequency targeted mutagenesis | Argonaute 18 (ZmAgo18a and ZmAgo18b), dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4) | Involved in sporogenesis and anthocyanin biosynthesis | Agrobacterium-mediated transformation | No | [48] |
Reduction of the linkage drag during breeding procedure | LG1 gene | Genetic basis for the upright architecture of maize leaves | Agrobacterium-mediated transformation of immature embryos // gene knockout with Cas9/gRNA | No | [49] | |
Biofortification | ||||||
Crops | ||||||
Camelina sativa | Enhancement of seed oil (fatty acid) composition in seeds | Fatty acid desaturase 2 (FAD2) genes | Key gene involved in the synthesis of polyunsaturated fatty acids [insertion of a double bond at the delta-12 (omega-6) position of oleic acid to obtain linoleic acid] | Agrobacterium-mediated transformation with Cas9/gRNA plasmid vectors (floral dipping) // gene knockout with Cas9/gRNA | No | [50] |
Reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil | Fatty acid desaturase 2 (FAD2) | Key gene involved in the synthesis of polyunsaturated fatty acids [insertion of a double bond at the delta-12 (omega-6) position of oleic acid to obtain linoleic acid] | Agrobacterium-mediated transformation with Cas9/gRNA plasmid vectors (floral dipping) // gene knockout with Cas9/gRNA | No | [51] | |
Seed oil biosynthesis | CsDGAT1 or CsPDAT1 homeologous genes | Involved in triacylglycerol (TAG) synthesis in developing seeds | Agrobacterium-mediated floral vacuum infiltration method // CRISPR–Cas9-mediated multiplex genome editing | No | [52] | |
Hordeum vulgare cv. “Golden Promise” | N-glycans modification in cereal grains | The putative endogenous barley ENGase gene | Involved in N-glycans biosynthesis | Co-bombarding selected combinations of sgRNA with wild-type cas9 using separate plasmids, or by co-infection with separate Agrobacterium tumefaciens cultures // CRISPR–Cas9-mediated multiplex genome editing | No | [53] |
Nicotiana tabacum | Production of biotherapeutic proteins | XylT gene FucT gene |
Involved in glycans biosynthesis | Agrobacterium-mediated transformation // gene knockout with Cas9/gRNA | No | [54] |
Production of biotherapeutic proteins | Beta(1,2)-xylosyltransferase (XylT) and alpha(1,3)fucosyltransferase (FucT). | Involved in glycans biosynthesis | Agrobacterium-mediated transformation // CRISPR-Cas9-mediated multiplex genome editing | No | [55] | |
Oryza sativa | Generation of high-amylose rice | SBEI and SBEIIb genes | Starch branching enzyme (SBE) genes involved in starch biosynthesis | Agrobacterium-mediated transformation // gene knockout with Cas9/gRNA | Yes | [56] |
Papaver somniferum | Biosynthesis of Benzylisoquinoline alkaloids (BIAs): medical biomolecules | 3′-hydroxyl-N-methylcoclaurine 4′-O-methyltransferase isoform 2 (4′ OMT2) gene | Implicated in the regulation of the biosythesis of benzylisoquinoline alkaloids (BIAs, e.g. morphine, thebaine) | Agrobacterium-mediated transformation of leaves with TRV-based synthetic plasmids expressing gRNA and a Cas9-encoding synthetic vector // gene knockout with Cas9/gRNA | No | [19] |
Solanum tuberosum | Starch quality (amylopectin potato starch) | Three different regions of the gene encoding granule-bound starch synthase (GBSS) | Enzyme responsible for the synthesis of amylose (encoded by a single locus) | PEG-mediated protoplast transfection with CRISPR-Cas9 expression plasmid constructs // gene knockout with Cas9/gRNA | Yes | [12] |
Salvia miltiorrhiza | Knock out the committed diterpene synthase gene | Diterpene synthase gene SmCPS1 | Involved in tanshinone biosynthesis | Agrobacterium rhizogenes-mediated transformation // gene knockout with Cas9/gRNA | No | [57] |
Conservation parameters | ||||||
Crops | ||||||
Solanum lycopersicum | Inhibition of tomato fruit ripening | Three regions within the RIN gene (ripening inhibitor) | Master regulator gene for tomato fruit ripening; encodes a MADS-box transcription factor regulating fruit ripening | Agrobacterium-mediated transformation with Cas9/sgRNA-expressing plasmid vectors // CRISPR–Cas9-mediated multiplex genome editing | Yes | [58] |