. 2017 Nov 10;1(2):169–182. doi: 10.1042/ETLS20170085

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]
Nicotiana benthamiana	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 (EBE_PthA4-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 EBE_PthA4-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]
Arabidopsis thaliana	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]
Zea mays	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]
Nicotiana tabacum	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]