1. Introduction

Agricultural Sciences

2156-8553

Scientific Research Publishing

10.4236/as.2019.1010097

AS-95813

Articles

Biomedical&Life Sciences Earth&Environmental Sciences

Targeted Genome Engineering and Its Application in Trait Improvement of Crop Plants

Xiaoyu

Yang

¹^*Linlin

Luo

²Beixin

²Lin

Liu

Guangdong Provincial Key Laboratory for Plant Epigenetics, Longhua Bioindustry and Innovation Research Institute, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China

Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, China

09102019

10101312134219, September 201915, October 2019 18, October 2019

2014

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Targeted genome engineering refers to technologies that are used for site-specific genome modifications such as knockout, knockin and transcriptional regulation of genes of interest in organisms. Site-specific recombination system, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9) technologies are the representatives of targeted genome engineering and have been widely used in crop basic and applied research. In this review, we introduce the basic information and action modes of these different genome engineering technologies, summarize the recent progresses of targeted genome engineering technologies and their applications in crop improvement, and propose perspectives for genome engineering-mediated modifications of crop plants in the future.

Targeted Genome Engineering Site-Specific Recombination ZFN TALEN CRISPR/Cas9 Crop Improvement

1. Introduction

In the past decades, global climate changes and improper agricultural practices have caused serious environmental problems such as soil salinization and acidification [1] , soil erosion [2] , drought [3] and explosion of insect pests [4] , which severely challenge agricultural production in the world, while simultaneously the increasing of global crop productivity is required to meet the growing demand for grain products from continuously increased world population [5] . It becomes an urgent task for current agricultural scientists to create novel crop varieties with better agronomic traits such as high yield and grain quality, high nutrient usage efficiency, and good acclimation capacity to abiotic and biotic stresses. Conventional breeding strategies such as genetic hybridization have been widely used for creation of new crop varieties and obtained tremendous successes for a long period, but these processes are commonly time-consuming and labor-intensive, thus being difficult to satisfy modern plant breeding objectives.

The rapid development of molecular biology and transgene technologies has made it feasible to genetically modify the traits of interest, and lots of transgenic plants harboring beneficial agronomic traits such as golden rice, a kind of GM rice that is able to accumulate β-carotene in grain, have been generated [6] [7] [8] . In contrast to the widespread utilization of transgenic technology in the basic and applied research of crops in the lab, commercialization of GM crops is still strictly regulated by governments in the world such as China, Japan and European countries because of intensive concerns about biosafety of GM crops [9] . Therefore, only a very small portion of GM crops have been released by now [10] [11] [12] [13] .

Targeted genome engineering refers to technologies that are used for site-specific genome modifications including gene knockout, knockin and transcriptional regulation [14] [15] . Site-specific recombination system, ZFNs, TALENs and CRISPR/Cas9 technologies are the representatives of targeted genome engineering. In comparison to the conventional transgenic technology, genome editing has obvious advantages such as easy design and construction, high precision and efficiency of modifications for genome loci responsible for traits of interest, being capable of stacking multiple genes of interest simultaneously, generation of descendants without transgenic elements, and so on. Therefore, targeted genome engineering has attracted extensive attentions from plant scientists and breeders, and has been rapidly adopted in crop improvement, especially with the emergence of CRISPR/Cas9 system since 2013 [16] [17] . In this review, we summarize recent progresses of targeted genome engineering and its application in genetical modifications of crop plants, and propose perspectives for future research on genome editing-based crop improvement.

2. Site-Specific Recombination System

Site-specific recombination refers to the reaction of two DNA molecules catalyzed by specific enzymes (recombinases) at their cognate pairs of sequences or target sites [18] . This recombination requires three components, a recombinase such as Cre responsible for DNA editing and two DNA partners such as LoxP cognate sites recognized by the recombinase. The recombinases can be divided into two families, tyrosine recombinase family and serine recombinase family (Table 1) [18] . The tyrosine recombinases contain a conserved tyrosine active site and catalyze DNA rearrangement via formation and resolution of a Holliday junction intermediate [19] , while the serine recombinases contain a conserved serine active site and catalyze site-specific DNA recombination through a concerted, four-strand cleavage and rejoining mechanism [20] .

Among these site-specific recombination systems, Cre/LoxP is the most commonly used one, which is composed of Cre recombinase and 34-bp LoxP sequences that can be recognized by Cre [21] . When the Cre recombinase is expressed, recombination events will occur to the cells harboring LoxP recognition sites in their genomes. In general, there are three possible outcomes from the Cre/LoxP-derived recombination including inversion, translocation or excision, depending on the initial arrangement of LoxP recombination sites (Figure 1). Inversion event or excision event can occur when the recombination sites are located on the same chromosome with the same orientation or the opposite orientation, respectively. Translocation event can result from the exchange of DNA segments when the two recombination sites are located on separate chromosomes with the same orientation [21] .

There are two main applications of site-specific recombination system in genetical modifications of crop plants: removal of undesirable transgenic elements such as selectable marker genes and site-specific integration of genes of interest. For example, by using Cre/LoxP-mediated recombination, the selectable maker gene, HPT, has been successfully eliminated from transgenic mustard plants with insect resistance [22] . Elimination of selectable marker genes has also been reported in rice [23] , potato [24] , tomato [25] and so on. Cre/LoxP system has been adopted in construction of maize and rice minichromosomes as well, wherein genes of interest are expected to be stacked without limitations [26] [27] . The application of Flp/Frt recombination system has also been reported for elimination of selectable marker genes in plants such as rice [28] and maize [29] . The representative GM crops that were generated via site-specific recombination recently are summarized in Table 2.

Although targeted genome editing has been realized via different site-specific recombination tools, there are still disadvantages limiting their applications in current crop improvement such as the failure of complete removal of transgenic elements, complicated design for vectors, time-consuming multiple transformation and genetic hybridization, and relatively low targeting efficiency [52] .

Table 1 Representative recombinases responsible for site-specific recombination [<xref ref-type="bibr" rid="scirp.95813-ref18">18</xref>]

Recombinase family	Representative
Tyrosine recombinase family	Cre, Flp, R, λInt, Int of Tn916/Tn1545, IntI, XerC/D, TnpI of Tn4430, FimB/E, Rci of R64, XisA/C
Serine recombinase family	Cin or Gin, Int of φC31/Bbv1/φRv1, TnpR of Tn3/γδ, Sin of S. aureus, ParA of RP4, Hin, OrfA of IS607/IS1535, TnpX of Tn4451, SpoIVCA, XisF

3. ZFN Technology

ZF proteins are the common group of DNA binding proteins in eukaryotic organisms. Each ZF protein is composed of about 30 amino acids in a conserved β-β-α configuration [53] . The DNA binding ability is determined by the specific amino acids present on the surface of the α-helix in each ZF with varying specificity. Based on the specific DNA binding trait, a targeted genome editing platform, ZFNs, has been constructed (Figure 2). A ZFN system is composed of two arrays of ZF proteins and a nuclease such as Fok I. Each array of ZF proteins is linked with a subunit of Fok I. Fok I can work normally after the two arrays of ZF proteins bind to the DNA sites of interest and two subunits of Fok I are dimerized. There are several strategies used for the assembly of ZFNs. The first one is called as modular assembly, a strategy based on the library of ZFs with well-known DNA-binding specificities. ZFNs can also be assembled through web-based tools by combining random assembly of multi-finger libraries with specificity screening or by companies.

In general, there are two types of DNA editing by ZFNs (Figure 2). The first type is the targeted gene knockout, of which the purpose is to create a null mutant by interfering with the expression of genes of interest at DNA level. For example, the HIV-1 resistance has been detected in the primary T cells and the hematopoietic stem/progenitor cells by ZFN-mediated knockout of the CC chemokine receptor 5 [54] [55] . A maize ipk1 mutant line has also been generated by ZFN-mediated gene knockout, leading to a modified phytate biosynthesis pathway in the resulted maize plants [56] . The other is the targeted gene

Table 2 The application of site-specific recombination system in targeted gene modifications of crop plants

Modification type	Crop	System	Target	Purpose	Reference
Removal of transgenic elements	Apple	Flp/Frt	NPTII, RVI6	Marker gene excision, scab resistance	[30]
	Barley	phiC31	GFP	Excision assessment	[31]
	Barley	Cre/LoxP	BAR	Marker gene excision	[32]
	Grapevine	Flp/Frt	NPTII	Marker gene excision	[33]
	Maize	Cre/LoxP	GAT, CFP	Excision assessment	[34]
	Maize	Cre/LoxP	GFP	Marker gene excision	[35]
	Mustard	Cre/LoxP	HPT	Marker gene excision	[22]
	Oilseed rape	Cre/LoxP	NPTII	Marker gene excision	[36]
	Orange	Cre/LoxP	AATCB, IPT	Canker resistance, marker gene excision	[37]
	Potato	Cre/LoxP	BAR	Marker gene excision	[38]
	Rice	Cre/LoxP	NOS terminator	Transgenic element excision	[39]
	Rice	Flp/Frt	HPT, TC	Enhancement of tocopherol content, marker gene excision	[40]
	Soybean	Cre/LoxP	NPTII	Marker gene excision	[41]
	Tobacco	Cre/LoxP	NPTII	Marker gene excision	[42]
	Tobacco	CinH-RS2	GFP	Excision assessment	[43]
	Tobacco	ParA-MRS, CinH-RS2	RFP	Marker gene excision	[44]
	Wheat	Cre/LoxP	BAR	Marker gene excision	[45]
Gene integration	Maize	Cre/LoxP	RFP	Insertion assessment	[26]
	Maize	Flp/Frt	PMI, RFP	Insertion assessment	[46]
	Oilseed rape	Cre/LoxP, Flp/Frt	RFP, GFP	Insertion assessment	[47]
	Rice	Cre/LoxP	GUS	Insertion assessment	[48]
	Rice	Cre/LoxP, ZFN	UBI promoter, clone sites for genes of interest	Insertion assessment	[49]
	Soybean	Flp/Frt	ALS, CFP	Insertion assessment	[50]
	Tobacco	R/Rs	LUC	Insertion assessment	[51]

knockin, of which the purpose is to create organisms expressing genes of interest by introducing exogenous genes at a specific site of the genome or by repairing the mutation sites of endogenous genes. In this case, for example, ZFNs have been applied to repair the mutation sites that are closely associated with diseases such as haemophilia B [57] , sickle-cell disease [58] and Parkinson’s disease [59] . In tobacco BY2 cells, a functional GFP gene has been successfully introduced into both the pre-integrated defective reporter construct and an endogenous locus

by ZFNs [60] . In addition to the targeted gene knockout and knockin, ZFs can be linked with transcriptional factors to regulate gene expression at DNA level [61] . Table 3 shows a summary of representative GM crops that were generated via ZFN system recently.

Despite the successes of ZF-associated technologies in previous studies, some disadvantages such as the complexity of assembly, context-dependent binding specificity and relatively low targeting efficiency have not been well addressed and thus limited the application of these technologies in current research [74] .

4. TALEN Technology

TALEs are a group of natural proteins from the genus of plant bacteria, Xanthomonas, and can bind some specific DNA regions in plant genome through a series of 33-35 amino acid domains that each can recognize a single base pair [75] . The binding specificity of TALEs is dependent on the RVD, the two highly-variable amino acids at the position of 12 and 13 in each TALE (Table 4) [75] . TALEs can be designed to recognize specific DNA sequences in the genome. By

Table 3 The application of zinc finger nucleases (ZFNs) or ZF transcription factors in targeted gene modifications of crop plants

Modification type	Crop	Target	Purpose	Reference
Knockout	Apple	GUS	Mutagenesis assessment	[62]
	Fig	GUS	Mutagenesis assessment	[62]
	Maize	IPK1	Alteration of inositol phosphate profile	[56]
	Poplar	LFY, AG	Complete sexual sterility	[63]
	Tobacco	SUR	Herbicide resistance	[64]
	Tobacco	GUS	Transgene disruption	[65]
	Soybean	DCL1a, DCL1b, DCL4a, DCL4b	Mutagenesis assessment	[66]
Knockin	Maize	PAT	Herbicide resistance	[56]
	Maize	AAD1	Herbicide resistance	[67]
	Maize	PAT	Herbicide resistance	[68]
	Maize	BAR	Herbicide resistance	[69]
	Rice	GUS	Identification of safe harbor loci	[70]
	Soybean	AAD1, DSM2, DGT28, HPTII, RPF	Multigene insertion assessment	[71]
	Tobacco	NPTII, RFP	Insertion assessment	[72]
Transcription activation	Oilseed rape	KASII	Improvement of oil production	[73]

Table 4 The corresponding nucleotide of amino acid pairs in transcriptional activator-like effector (TALE) [<xref ref-type="bibr" rid="scirp.95813-ref75">75</xref>]

RVD in TALE	The corresponding nucleotide
NI (Asparagine and Isoleucine)	A
HD (Histidine and Aspartate)	C
NN (Asparagine and Asparagine)	G/A
NG (Asparagine and Glycine)	T

adding a nuclease such as the Fok-I, TALEN system has been developed to make DSBs on pre-selected genome sites to generate knockout or knockin editing in the genome (Figure 3).

For example, AvrXa7 is the effector-binding element in the promoter of a bacterial blight susceptibility gene Os11N3, and the TALEN-mediated knockout of this element has generated rice with resistance to bacterial blight [76] . In another report, rice fragrance has been improved by TALEN-mediated targeting to a defective badh2 allele, which is responsible for the synthesis of 2AP, a major fragrance compound in rice [77] . The TALE designer transcription factors have been used to regulate OCT4 and NANOG loci by targeting their enhancers in mammalian cells, leading to stimulation or inhibition of reprogramming somatic cells to the induced pluripotent cells [78] , while similar applications have not been introduced into plants yet. Table 5 shows a summary of representative GM crops that were generated via TALEN recently.

The single base recognition of TALE-DNA binding repeats affords greater design flexibility than triplet-confined ZF proteins, while the assembly of the large repetitive TALE modules is still a great challenge for the cloning of repeated TALE arrays [74] .

5. CRISPR/Cas9 System

CRISPR/Cas is an adaptive innate immune system to defend against the invasion of viral and plasmid DNA in bacteria and archaes [99] . Scientists have found three types of CRISPR/Cas systems (type I, II and III) within a series of microbes, and each type includes a Cas protein and the corresponding CRISPR arrays [100] . These CRISPR/Cas arrays are composed of repeat sequences interspaced by spacer sequences, which are non-repetitive and derive from the short segments of viral or plasmid DNA often called as protospacer sequences. By transcribing the CRISPR arrays into crRNAs, Cas proteins can be directed to the target sites and make DSBs, resulting in the degradation of foreign genetic materials.

Table 5 The application of transcription activator-like effector nuclease (TALEN) technology in targeted gene modifications of crop plants

Modification type	Crop	Target	Purpose	Reference
Knockout	Brassica oleracea	FRI	Mutagenesis assessment	[79]
	Maize	GL2	Alteration of wax compositions	[80]
	Maize	MTL	Haploid induction	[81]
	Peanut	FAD2	Alteration of lipid compositions	[82]
	Potato	SSR2	Inhibition of cholesterol synthesis	[83]
	Potato	ALS1	Mutagenesis assessment	[84]
	Potato	VINV	Reduction of acrylamide content	[85]
	Potato	SBE1, VINV	Alterations of starch compositions and cold sweetening trait	[86]
	Rice	11N3	Bacterial blight resistance	[76]
	Rice	BADH2	Improvement of grain fragrance	[77]
	Rice	11N3	Bacterial blight resistance	[87]
	Sugarcane	COMT	Alteration of cell wall composition	[88]
	Sugarcane	COMT	Alteration of cell wall composition	[89]
	Soybean	FAD2-1a, FAD2-1b	Alteration of lipid compositions	[90]
	Soybean	FAD2-1a, FAD2-1b, FAD3a	Alteration of lipid compositions	[91]
	Tobacco	ALS	Mutagenesis assessment	[92]
	Wheat	MLO	Powdery mildew resistance	[93]
Knockin	Barley	GFP	Insertion assessment	[94]
	Potato	ALS1	Herbicide resistance	[95]
	Potato	ALS, NPTII	Insertion assessment, herbicide resistance	[96]
	Rice	ALS	Herbicide resistance	[97]
	Tobacco	YFP	Insertion assessment	[92]
	Tomato	35S promoter	Anthocyanin accumulation	[98]

Among the three CRISPR systems, the type II CRISPR system is the first one to be engineered for targeted genome editing in eukaryotic organisms (Figure 4). In this system DNA sequence bearing a 5’-NGG-3’ PAM can be recognized by a duplex of two non-coding RNAs, a crRNA and a tracrRNA, or with a sgRNA, which is a synthetic fusion of crRNA and tracrRNA, and then be degraded by Cas9 protein that is complexed with the duplex of crRNA and tracrRNA or the sgRNA [16] [17] . The Cas9 protein from Streptococcus pyogenes (SpCas9) has two nuclease motifs, HNH and RuvC, which play critical roles in generating DSBs at target sites [101] . The target specificity of CRISPR/Cas9 is determined by a seed sequence, which is a 12-base sequence upstream of the PAM and must match with the sequence of crRNA or sgRNA [102] . Compared with the

formerly-designed ZFNs and TALENs, CRISPR/Cas9 system has more advantages such as the simplicity of design and assembly, the high efficiency of targeting and the versatility of application, and thus is expected to be a powerful tool for targeted genome editing (Table 6).

Table 6 Characteristic comparison of different targeted genome engineering systems

	Cre/LoxP	Flp/Frt	ZFN	TALEN	CRISPR/Cas9
Design and assembly	Easy	Easy	Difficult	Difficult	Easy
Targeting site	34 bp	34 bp	9 - 18 bp for ZFN monomer, 18-36 bp for ZFN dimer	14 - 20 bp for TALEN dimer, 28 - 40 bp for TALEN dimer	23 bp including 20 bp binding site and 3 bp PAM, multiple editing simultaneously
Restriction for target sites on genome	High	High	No	No	Low
Mismatch tolerance at target site	No	No	Low	Low	Relatively high
Targeting efficiency	Low	Low	Low	Moderate	High
Repair mechanism	HR	HR	NHEJ and HR	NHEJ and HR	NHEJ and HR
Off-target effects	Not available	Not available	Low	Low	Low
Transgene-free in offspring	No	No	Yes	Yes	Yes

CRISPR/Cas9 is first reported to be efficient in targeted genome editing in mammalian cells at single-gene level [16] [17] , multi-gene level [103] and even genome-wide level [104] . Thereafter, this system is adopted for gene knockout or knockin in plants such as Arabidopsis, tobacco, sorghum, rice, and wheat [105] [106] . By modifying two amino acids at the position of 10 and 841 from aspartate (D) and histidine (H) to alanine (A), scientists have successfully developed two variants of Cas9, Cas9n (D10A) with a nickase activity and dCas9 (D10A and H841A) without any catalytic activity. The two variants of Cas9 have been further developed to CRIPSR/Cas9n [107] and CRISPR/dCas9 systems [108] . Some Cas9-derived base editor systems have also been established by fusing an adenine or a cytidine deaminase to a Cas9n protein [109] . Being different from the classical Cas9 system, the base editor systems can catalyze two kinds of nucleotide replacement reactions, from adenine (A) to guanine (G) or from cytosine (C) to thymine (T), depending on the deaminase that is linked to Cas9n [109] . Applications of these Cas9-derived systems have been reported in targeted genome editing. For example, in a comparison study of gene expression regulation by CRISPR/dCas9 and TALE designer transcription factor systems, scientists have demonstrated better performance in activation of gene expression by TALE activator system, while repression by CRISPR/dCa9 was similar with or better than TALE repressor [78] . The successful utilization of base editors has been well documented in crop plants such as rice, tomato, potato, wheat, maize, rape and watermelon as well [110] - [118] . To date, CRISPR/Cas9-mediated genome editing has been widely used for crop improvement and a number of GM crops with desired traits have been created (Table 7).

Recently, some orthologues of the classical Cas9 (SpCas9) have been identified in bacteria such as SaCas9 from Staphylococcus aureus and St1Cas9 from Streptococcus thermophilus [154] , and been adopted in targeted genome modifications

Table 7 The application of CRISPR/Cas9 in targeted gene modifications of crop plants

Modification type	Crop	Target	Purpose	Reference
Knockout	Apple	DIPM1, DIPM2, DIPM3, DIPM4	Fire blight disease resistance	[119]
	Apple	PDS	Mutagenesis assessment	[120]
	Banana	PDS	Mutagenesis assessment	[121]
	Camelina	FAD2	Alteration of lipid compositions	[122]
	Cassava	PDS	Mutagenesis assessment	[123]
	Cassava	nCBP-1, nCBP-2	Brown streak disease resistance	[124]
	Citrus	LOB1	Citrus canker resistance	[125]
	Cucumber	eIF4E	Virus resistance	[126]
	Flax	EPSPS	Herbicide resistance	[95]
	Grape	IdnDH	Improvement of tartaric acid production	[127]
	Oilseed rape	FAD2	Alteration of lipid compositions	[128]
	Potato	ALS1	Herbicide resistance	[129]
	Potato	GBSS	Alteration of starch content	[130]
	Potato	MYB44	Improvement of phosphate usage efficiency	[131]
	Potato	S-Rnase	Improvement of self-compatibility	[132]
	Rice	ERF922	Blast resistance	[133]
	Rice	HAK1	Inhibition of Cs⁺ accumulation	[134]
	Rice	CYP97A4, DSM2, CCD4a, CCD4b, CCD7	β-carotene accumulation, plant architecture	[135]
	Tobacco	PDS, PDR6	Optimization of plant architecture and yield	[136]
	Tomato	MAPK3	Drought resistance	[137]
	Wheat	GASR7, DEP1	Enhancement of thousand grain weight	[138]
	Wheat	ABCC6, NFXL1, LTP9.4	Fusarium head blight disease resistance	[139]
	Wheat	DREB2, ERF3	Drought resistance, ethylene responses	[140]
	Wheat	GW2	Enhancement of seed size and thousand grain weight	[141]
Knockin	Flax	BFP	Insertion assessment	[129]
	Maize	ALS2, PAT	Chlorsulfuron resistance, herbicide resistance	[142]
	Rice	TB-TIPS-E2 fragment, E2-TIPS-E8 fragment	Insertion assessment	[143]
	Rice	HPTII, RFP	Insertion assessment	[144]
	Soybean	HPT	Insertion assessment	[145]

Knockin	Tobacco	AvrII recognition site	Insertion assessment	[146]
	Tomato	ALS2	Chlorsulfuron resistance	[147]
	Wheat	GFP	Insertion assessment	[148]
Transcriptional activation	Rice	GW7, ER1	Activation assessment	[149]
	Rice	Os03g01240, Os04g39780, Os11g35410	Activation assessment	[150]
	Tobacco	PDS	Activation assessment	[151]
	Tobacco	GUS	Activation assessment	[152]
	Tobacco	LUC	Activation assessment	[153]
Transcriptional repression	Tobacco	PDS	Repression assessment	[151]
Transcriptional repression	Tobacco	LUC	Repression assessment	[153]

of Arabidopsis, tobacco and rice (Table 8) [155] [156] . The comparable target efficiency of SaCas9 and St1Cas9 with SpCas9 system has also been documented in plants [155] . Although all of the three Cas9 proteins are clustered to the type II CRISPR/Cas system, different PAM sequences are recognized (5’-NNGRRT-3’ for SaCas9 and 5’-NNGGAA-3’ for St1Cas9 instead of 5’-NGG-3’ for SpCas9) when DSBs are catalyzed at the target sites [157] . Considering that the flexibility of target sites is constrained by PAM sequences to great extent, the introduction of SaCas9 and St1Cas9 into CRIPSR group greatly expands the coverage of available target sites on plant genome and promotes CRISPR-mediated crop improvement. In addition to the mentioned type II CRISPR systems above, a CRISPR/Cpf1 platform (also named CRISPR/Cas12a) has been developed, of which several aspects are quite distinct from the Cas9 systems [158] [159] . For instance, only a crRNA is needed for guiding Cpf1-crRNA complex to target sites on plant genome, and therefore, the length of sgRNA is shortened from approximately 100 nt in Cas9 systems to 42 nt, facilitating sgRNA synthesis. The PAM sequence of Cpf1 is 5’-TTTN-3’ and the Cpf1-mediated DSBs can generate 4 bp overhangs at 5’ end of cleavage sites, while blunt ends are produced after Cas9 cleavage at target sites. The resulted cohesive ends by Cpf1 display some potential advantages over blunt ends such as the improvement of knockin efficiency and the increased possibility of multiple editing events. The CRISPR/Cpf1 system is thus considered as a promising tool and has been applied in genetical modifications of crop plants [Table 8]. And we believe that this system will be widely used for crop improvement in the near future.

6. Perspectives

The appearance and rapid development of targeted genome engineering technologies have paved a new way for crop breeding. By now, four generations of genome editing platforms have been provided from site-specific recombination system to ZFN, TALEN and CRISPR/Cas9 technologies. CRISPR/Cas9 is now

Table 8 The newly-developed CRISPR systems and their applications in crop improvement

System	Source	Crop	Target	Purpose	Reference
CRISPR/FnCas9	Francisella novicida	Tobacco	CMV and TMV genomes	Virus resistance	[160]
CRISPR/SaCas9	Staphylococcus aureus	Rice	DL	Mutagenesis assessment	[156]
CRISPR/SaCas9	Staphylococcus aureus	Tobacco	PDS, FT4	Mutagenesis assessment	[156]
CRISPR/LshCas13a	Leptotrichia shahii	Rice	SRBSDV genome	Virus resistance	[161]
CRISPR/LshCas13a	Leptotrichia shahii	Tobacco	TMV genome	Virus resistance	[161]
CRISPR/Cpf1	Acidaminococcus sp. BV3L6	Rice	ROC5, DEP1	Temperature-dependent target efficiency improvement	[162]
CRISPR/Cpf1	Francisella novicida	Rice	DL, ALS	Leaf morphology	[163]
CRISPR/Cpf1	Francisella novicida	Tobacco	PDS, STF1	Leaf morphology and pigment biosynthesis	[163]
CRISPR/Cpf1	Lachnospiraceae bacterium	Maize	GL2	Target specificity assessment	[164]
CRISPR/Cpf1	Lachnospiraceae bacterium	Maize	GL2	Temperature-dependent target efficiency improvement	[162]
CRISPR/Cpf1	Lachnospiraceae bacterium	Rice	DEP1, SPL14, TB1, LAC, MIR398a, Os02cir25329, Os03cir00204, PDS, ROC5	Target specificity assessment	[165]
CRISPR/Cpf1	Lachnospiraceae bacterium	Rice	BEL-230, BEL-240, BEL-250, BEL-260, EPSPS, RLK-798, RLK-799, RLK-802, RLK-803	Multigene mutagenesis assessment	[166]
CRISPR/Cpf1	Lachnospiraceae bacterium	Rice	EPFL9	Stomata development	[167]

considered as the advantageous tool over the other three and has been used in genetical modifications of crops most widely. A lot of new crop germplasms that do not exist in nature have been efficiently created via CRISPR-mediated knockout, knockin, transcriptional activation and transcriptional repression of genes of interest.

However, challenges still remain in current CRISPR platform. Thus far, most of reported modifications for crops are CRISPR-mediated gene knockouts while knockin events are rare and usually at low efficiency, though knockin is very useful for crop breeding because it can confer novel traits that do not exist in crops in nature by editing of existing alleles or adding of new ones. CRISPR/dCas9 is considered as a promising tool for transcriptional regulation of gene expression, particularly for genes with highly methylated promoter regions [168] [169] , while available data for gene modification of this kind are quite limited, constraining the improvement of CRISPR-based transcriptional regulation systems and their applications in crops. Transformation and tissue culture are crucial for CRISPR-mediated genome editing, while their efficiencies are challenged for most of crop plants. Last but not the least, off-targeting is still an intensive concern for plant scientists, though numerous studies have evidenced the precision of CRISPR-mediated genome editing in plants [170] . All of these challenges should be addressed in the future studies in order to promote the applications of CRISPR systems in crop improvement.

Acknowledgements

This work was supported by Natural Science Foundation of Guangdong Province (2018A030310446), China Postdoctoral Science Foundation (2017M612741), Guangdong Innovation Research Team Fund (2014ZT05S078), National Natural Science Foundation of China (31600982), and Shenzhen High-Level Talents Research Fund (827/000256).

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

Cite this paper

Yang, X.Y., Luo, L.L., Mo, B.X. and Liu, L. (2019) Targeted Genome Engineering and Its Application in Trait Improvement of Crop Plants. Agricultural Sciences, 10, 1312-1342. https://doi.org/10.4236/as.2019.1010097

Abbreviations

11N3: 11 nodulin 3

2AP: 2-acetyl-1-pyrroline

AAD1: aryloxyalkanoate dioxygenase 1

AATCB: cecropin B gene with a signal peptide sequence AAT from the a-1-antitrypsin

ABCC6: ATP-binding cassette sub-family C member 6

AG: agamous gene

ALS1: acetolactate synthase 1

BADH2: betaine aldehyde dehydrogenase 2

BAR: phosphinothricin acetyl transferase

BEL-230: bentazon sensitive lethal 230

BFP: blue fluorescent protein

Cas9: CRISPR-associated protein9 nuclease

CCD4a: carotenoid cleavage dioxygenase 4a

CFP: cyan fluorescent protein

COMT: caffeic acid O-methyltransferase

Cre: cyclization recombination enzyme

CRISPR: clustered regularly interspaced short palindromic repeats

crRNA: CRISPR RNA

CYP97A4: cytochrome P450-type carotenoid hydroxylases 97A4

DCL1a: dicer-like 1a

DEP1: dense and erect panicle 1

DGT28: dow glyphosate tolerance

DIPM1: DspA/E-interacting protein of Malus × domestica 1

DL: drooping leaf

DREB2: DRE-binding protein 1

DSB: double-strand breakage

DSM2: drought-hypersensitive mutant 2

eIF4E: eukaryotic initiation factor 4E

EPFL9: epidermal patterning factor like 9

EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase

ER1: Erysiphe pisi resistance 1

ERF922: ethylene responsive factor 922

FAD2: oleate desaturase 2

Fim: fimbrial subunit

Flp: yeast 2-micron circle plasmid recombinase

FRI: FRIGIDA

Frt: Flp-recognition target

FT4: flowering locus T 4

GASR7: gibberellic acid-stimulated transcript-related 7

GAT: glyphosate acetyltransferase

GBSS: granule-bound starch synthase

GFP: green fluorescent protein

GL2: GLABRA 2

GM: genetically modified

GUS: β-glucuronidase

GW2: grain width 2

HAK1: high-affinity potassium transporter 1

HIV-1: human immunodeficiency virus type 1

HPT: hygromycin phosphotransferase

HR: homologous recombination

IdnDH: idonate dehydrogenase

Int: integrase

IPK1: inositol 1,3,4,5,6-pentakisphosphate 2-kinase

IPT: isopentenyl transferase

KASII: 3-ketoacyl-ACP synthase II

LFY: leafy

LOB1: lateral organ boundaries

LoxP: integration/crossover site on phage P1 genome

LTP9.4: lipid transfer protein 9.4

LUC: luciferase

MAPK3: mitogen-activated protein kinase 3

MLO: mildew resistance locus O

MTL: MATRILINEAL phospholipase

MYB44: v-myb avian myeloblastosis viral oncogene homolog 44

NANOG: Nanog homeobox

nCBP-1: novel cap binding protein 1

NFXL1: nuclear factor-X like 1

NHEJ: non-homologous end joining

NOS: nopaline synthase

NPTII: neomycin phosphotransferase II

OCT4: octamer-binding transcription factor 4

PAM: protospacer-adjacent motif

PAT: hosphinothricin acetyltransferase

PDR6: pleiotropic drug resistance 6

PDS: phytoene desaturase

PMI: phosphomannose isomerase

RFP: red fluorescent protein

RLK-798: receptor-like kinases 798

ROC5: rice outermost cell-specific gene 5

RVD: repeat-variable di-residues

RVI6: resistance to Venturia inaequalis 6

SBE1: starch-branching enzyme 1

sgRNA: single guide RNA

SPL14: SQUAMOSA promoter binding protein-like 14

SSR2: sterol side chain reductase 2

STF1: soybean TGACG-motif binding factor 1

SUR: sulfonylurea receptor

TALE: transcriptional activator-like effector

TALEN: transcriptional activator-like effector nuclease

TB1: teosinte branched 1

TC: tocopherol cyclase

tracrRNA: trans-activating crRNA

VINV: vacuolar invertase

Xis: excisionase

YFP: yellow fluorescent protein

ZF: zinc finger

ZFN: zinc finger nuclease

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