Table 1 Agricultural applications of the use of CRISPR systems in the 52 articles studied (2014–2017)
Plant speciesApplication perspectivesTargeted sequence(s)Molecular functionsDelivery method//main strategyTransgene-free plants studied (yes/no)Reference
BIOTIC STRESS TOLERANCE
Virus stress tolerance
Model plants
Arabidopsis thalianaPotyvirus resistance (TuMV)eIF(iso)4E, member of the eukaryotic translation initiation factorRecessive resistance alleles against various potyvirusesAgrobacterium-mediated transformation with a Cas9/gRNA recombinant plasmid binary vector (floral dipping) // gene knockout with Cas9/gRNAYes[9]
Arabidopsis thaliana and Nicotiana benthamianaBeet severe curly top virus (BSCTV) tolerance43 candidate sites in coding or non-coding sequences of the BSCTV genome for transient expression assays and selection of two sites for transgenic lines inductionVirus replication mechanismAgrobacterium-mediated transformation of leaves with Cas9/gRNA expression plasmid vectors // gene knockout with Cas9/gRNANo[7]
Nicotiana benthamianaTomato yellow leaf curl virus (TYLCV) resistanceCoding and non-coding sequences of TYLCVVirus replication mechanismAgrobacterium-mediated transformation of leaves with a TRV RNA replicon for the delivery of gRNAs into Cas9 overexpressing plants // gene knockout with Cas9/gRNANo[14]
Virus toleranceAGO2 geneContribution 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/gRNANo[20]
Crops
Cucumis sativusIpomovirus immunity, tolerance to the Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-WeIF4E (eukaryotic translation initiation factors 4E)Host factors for RNA viruses, recessive resistance alleles against virusesAgrobacterium-mediated transformation of cut cotyledons (without embryo) with binary vector containing Cas9/gRNA // gene knockout with Cas9/gRNAYes[21]
Fungus stress tolerance
Crops
Oriza sativaBlast (caused by Magnaporthe oryzae) toleranceEthylene responsive factor ERF transcription factor gene OsERF922Involved in the modulation of multiple stress toleranceAgrobacterium-mediated transformation of embryogenic calli with Cas9/gRNA-expressing binary vectors // single and multiplex gene knockout with Cas9/gRNAYes[22]
Solanum lycopersicumPowdery mildew resistanceSlMlo geneMajor contributor to powdery mildew susceptibilityAgrobacterium-mediated transformation of cotyledons with Cas9/gRNA expression plasmid vectors // gene knockout with Cas9/gRNAYes[23]
Triticum aestivumPowdery mildew (Blumeria graminis f. sp. Tritici) resistanceOne of the three mildew-resistance locus (MLO) homeologs in bread wheat: TaMLO-A1 alleleEncode a protein that was shown to repress defenses against powdery mildew diseasesParticle bombardment with Cas9/gRNA expressing plasmid into immature wheat embryos // gene knockout with Cas9/gRNAYes[6]
Bacteria stress tolerance
Crops
Citrus paradisiCitrus canker (caused by Xanthomonas citri subspecies citri (Xcc)) tolerancePthA4 effector binding elements (EBEs) in the Type I CsLOB1 promoter (EBEPthA4-CsLOBP) of the CsLOB1 (Citrus sinensis lateral organ boundaries) geneCsLOB1: susceptibility gene for citrus canker CsLOB1 gene expression induced by the binding of the pathogenicity factor PthA4 to the EBEPthA4-CsLOBPAgrobacterium-mediated transformation of epicotyl with Cas9/gRNA expression plasmid vectors // gene knockout with Cas9/gRNANo[24]
Citrus sinensis OsbeckCanker resistanceCsLOB1 promoterSusceptibility gene CsLOB1 promoter in citrusAgrobacterium-mediated epicotyl transformation // gene knockout with Cas9/gRNANo[25]
Oryza sativaBacterial blight (caused by Xanthomonas oryzae pv. oyzae) toleranceSucrose transporter gene OsSWEET13Disease-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/gRNANo[26]
ABIOTIC STRESS TOLERANCE
Herbicide tolerance
Model plants
Arabidopsis thalianaCold, salt and drought stress toleranceUDP-glycosyltransferases UGT79B2 and UGT79B3UGT 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 stressesAgrobacterium-mediated transformation with a Cas9/gRNA recombinant plasmid binary vector via floral dipping // gene knockout with Cas9/gRNANo[27]
Glufosinate resistance and reduced trichomes formationBAR 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/gRNAYes[28]
Lotus japonicusBioavailability of soil organic nitrogen and capability to accommodate nitrogen-fixing bacteria intracellularly to fix its own nitrogenSingle and multiple symbiotic nitrogen fixation (SNF) genes: simbiosis receptor-like kinase (SYMRK), leghemglobin loci (LjLb1, LjLb2, LjLb3)Involved in symbiotic nitrogen fixationA. tumefaciens and A. rhizogenes-mediated transformation containing the appropriate plasmids // gene knockout with Cas9/gRNANo[3]
Crops
Linum usitatissimumGlyphosate tolerance5′-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 phosphoenolpyruvateProtoplast transfection with ssODN and CRISPR-Cas9 plasmid // gene replacementNo[15]
Oryza sativaHerbicide resistanceC287 geneThe C287Tgene mutation endows rice plants with resistance to the herbicide imazamox (IMZ)Agrobacterium-mediated transformation // CRISPR-Cas9-mediated multiplex genome editingNo[29]
Herbicide toleranceAcetolactate synthase (ALS) geneInvolved 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 templateNo[16]
Glyphosate tolerance5′-Enolpyruvylshikimate-3-phosphate synthase (EPSPS)Involved in the biosynthesis of aromatic amino acidsCo-transformation of rice calli through particle bombardment with Cas9/gRNA expression plasmid and donor plasmid // gene insertion and replacement with a donor templateYes[30]
Solanum tuberosumReduced susceptibility to ALS-inhibiting herbicidesAcetolactate 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 replacementNo[18]
Salt stress tolerance
Crops
Oryza sativaSalt stress toleranceGT-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 salinityAgrobacterium-mediated transformation of leaves with Cas9gRNA plasmid expression vector // gene knockout with Cas9/gRNANo[1]
Drought stress tolerance
Crops
Zea maysImproved grain yield under field drought stress conditionsARGOS8Negative regulator of ethylene responsesCo-transformation of immature embryos by particle bombardment with DNA repair template Cas9-sgRNA expression plasmids // gene insertion or replacement with a donor templateNo[17]
YIELD, BIOFORTIFICATION AND CONSERVATION PARAMETERS
Yield
Crops
Brassica oleracea and Hordeum vulgarePod shatter and control of dormancyHvPM19
BolC.GA4.a
Positive regulator of grain dormancy
Involved in pod valve margin development
Agrobacterium-mediated transformation // gene knockout with Cas9/gRNAYes[31]
Dendrobium officinaleLignocellulose biosynthesisC3H, C4H, 4CL, CCR and IRX genesTarget genes are involved in the lignocellulose biosynthesis pathwayAgrobacterium-mediated transformation // gene knockout with Cas9/gRNANo[32]
Nicotiana tabacum, sylvestris and tomentosiformisRegulation of axillary bud growthNtPIN4 geneInvolved in auxin biosynthesisAgrobacterium-mediated transformation of leaves // gene knockout with Cas9/gRNAYes[33]
Oryza sativaStarch synthesis pathway in rice pollenPlastific large subunit of ADP-glucose pyrophosphorylase (OsAGPL4)Involved in the starch synthesis pathwayAgrobacterium-mediated transformation with Cas9/gRNA plasmid expression vector // gene knockout with Cas9/gRNANo[34]
Regulation of pollen tube growth and integrityRice 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 interactionAgrobacterium-mediated transformation of embryo-derived rice callus with Cas9/gRNA expression plasmids // gene knockout with Cas9/gRNANo[35]
Grain yield performanceGrain size3 (GS3) and Grain number 1a (Gn1a)Grain yield QTLs identified to regulate grain size and grain numberAgrobacterium-mediated transformation with Cas9/gRNA plasmid expression vector // gene knockout with Cas9/gRNAYes[36]
Grain weightGrain width 2 (GW2), grain width 5 (GW5) and thousand-grain weight (TGW6)Three major genes that negatively regulate rice grain weightAgrobacterium-mediated transformation with Cas9/gRNAs plasmid expression vector // CRISPR–Cas9-mediated multiplex genome editingYes[37]
Development of japonica photo-sensitive genic male sterile rice linesCarbon starved anther (CSA)One important locus for regulating photoperiod-controlled male sterility in japonica riceAgrobacterium-mediated transformation with two plasmids into calli // gene knockout with Cas9/gRNAYes[38]
Enhanced grain number, dense erect panicles, larger grain sizeCytokinin 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 architectureAgrobacterium-mediated transformation with Cas9/gRNA plasmid expression vectors // gene knockout with Cas9/gRNAYes[39]
Maintenance and determinacy of the flower meristemFLORAL ORGAN NUMBER2 (FON2) gene
OsMADS3 gene
Involved in meristem maintenance and in stamen specificationAgrobacterium-mediated transformation of calli // gene knockout with Cas9/gRNANo[40]
Rice caryopsis developmentOsSWEET11 geneSugar transporterAgrobacterium-mediated transformation of leaves // gene knockout with Cas9/gRNANo[41]
Stomatal developmentalEPFL9 genePositive regulator of stomatal developmental pathwayAgrobacterium-mediated transformation of immature embryos // gene knockout with CRISPR–Cas9/Cpf1 systemYes[42]
Developing marker-free transgenic plantsGUS geneMarker geneAgrobacterium or gene gun with a construct expressing Cas9 and two gRNAs // gene knockout with Cas9/gRNANo[43]
Rice developmentMPK1 and MPK6 gnesEssential genes for rice developmentAgrobacterium-mediated transformation of rice calli // gene knockout with Cas9/gRNAYes[5]
Regulation of seed developmentMEGs and PEGs genesInvolved in the regulation of nutrient metabolism and endosperm developmentAgrobacterium-mediated transformation // gene knockout with Cas9/gRNANo[44]
Breeding of early-maturing rice cultivarsHd2, Hd4 and Hd5 genesFlowering suppressors in Ehd1-dependent photoperiodic flowering pathway and major genes that negatively control the heading date of rice varieties grown in the north of ChinaAgrobacterium-mediated transformation // gene knockout with Cas9/gRNANo[45]
Solanum lycopersicumGeneration of parthenocarpic tomato plantsSlIAA9 geneA key gene controlling parthenocarpyAgrobacterium-mediated transformation of leaves // gene knockout with Cas9/gRNANo[46]
Taraxacum kok-saghyzRubber biosynthesis in hairy rootsTK 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/gRNANo[47]
Zea maysHigh-frequency targeted mutagenesisArgonaute 18 (ZmAgo18a and ZmAgo18b), dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4)Involved in sporogenesis and anthocyanin biosynthesisAgrobacterium-mediated transformationNo[48]
Reduction of the linkage drag during breeding procedureLG1 geneGenetic basis for the upright architecture of maize leavesAgrobacterium-mediated transformation of immature embryos // gene knockout with Cas9/gRNANo[49]
Biofortification
Crops
Camelina sativaEnhancement of seed oil (fatty acid) composition in seedsFatty acid desaturase 2 (FAD2) genesKey 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/gRNANo[50]
Reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oilFatty 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/gRNANo[51]
Seed oil biosynthesisCsDGAT1 or CsPDAT1 homeologous genesInvolved in triacylglycerol (TAG) synthesis in developing seedsAgrobacterium-mediated floral vacuum infiltration method // CRISPR–Cas9-mediated multiplex genome editingNo[52]
Hordeum vulgare cv.Golden PromiseN-glycans modification in cereal grainsThe putative endogenous barley ENGase geneInvolved in N-glycans biosynthesisCo-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 editingNo[53]
Nicotiana tabacumProduction of biotherapeutic proteinsXylT gene
FucT gene
Involved in glycans biosynthesisAgrobacterium-mediated transformation // gene knockout with Cas9/gRNANo[54]
Production of biotherapeutic proteinsBeta(1,2)-xylosyltransferase (XylT) and alpha(1,3)fucosyltransferase (FucT).Involved in glycans biosynthesisAgrobacterium-mediated transformation // CRISPR-Cas9-mediated multiplex genome editingNo[55]
Oryza sativaGeneration of high-amylose riceSBEI and SBEIIb genesStarch branching enzyme (SBE) genes involved in starch biosynthesisAgrobacterium-mediated transformation // gene knockout with Cas9/gRNAYes[56]
Papaver somniferumBiosynthesis of Benzylisoquinoline alkaloids (BIAs): medical biomolecules3-hydroxyl-N-methylcoclaurine 4-O-methyltransferase isoform 2 (4OMT2) geneImplicated 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/gRNANo[19]
Solanum tuberosumStarch 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/gRNAYes[12]
Salvia miltiorrhizaKnock out the committed diterpene synthase geneDiterpene synthase gene SmCPS1Involved in tanshinone biosynthesisAgrobacterium rhizogenes-mediated transformation // gene knockout with Cas9/gRNANo[57]
Conservation parameters
Crops
Solanum lycopersicumInhibition of tomato fruit ripeningThree regions within the RIN gene (ripening inhibitor)Master regulator gene for tomato fruit ripening; encodes a MADS-box transcription factor regulating fruit ripeningAgrobacterium-mediated transformation with Cas9/sgRNA-expressing plasmid vectors // CRISPR–Cas9-mediated multiplex genome editingYes[58]