Qmr U.Zmn,Cho Li,Hongto Cheng,Qiong Hu,*

a Oil Crops Research Institute,Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture,Wuhan 430062,Hubei,China

b Graduate School of Chinese Academy of Agricultural Sciences,Beijing 100081,China

Keywords:Genome editing CRISPR Site-specific mutagenesis Polyploid Crop improvement

A B S T R A C T Sequence-specific nucleases(SSN)that generate double-stranded DNA breaks(DSBs)in genes of interest are the key to site-specific genome editing in plants.Genome editing has developed into one method of reducing undesirable traits in crops by the induction of knockout mutations.Different SSN-mediated genome-editing systems,including LAGLIDADG homing endonucleases or meganucleases,zinc-finger nucleases,transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats,are emerging as robust tools for introducing functional mutations in polyploid crops including citrus,wheat,cotton,soybean,rapeseed,potato,grapes,Camelina sativa,dandelion,and tobacco.The approach utilizes knowledge of biological mechanisms for targeted induction of DSBs and their error-prone repair,allowing highly specific changes at designated genome loci.In this review,we briefly describe genome-editing technologies and their application to genetic improvement of polyploid crops.

1.Introduction

Polyploidy is a condition in which a diploid organism acquires one or more additional complete sets of chromosomes.There are three general categories of polyploid:autopolyploids,allopolyploids,and segmental allopolyploids.In autopolyploids,genome duplication occurs within a species(intraspecifically)so that all genomes are identical.Allopolyploids contain distinct genomes resulting from hybridization of two different species[1].Segmental allopolyploids consist of more than two partially differentiated genomes,which form bivalents and multivalents during chromosome pairing[2].Polyploidy plays a major role in speciation[3]and evolution[2].Polyploid crop genomes with multiple sets of chromosomes always undergo drastic changes in structure and function as a result of genetic and epigenetic changes[4].The large number of orthologous genes,heterozygosity,repetitive DNA,and genome irregularity of polyploid genomes pose challenges to genome mutation techniques.Several techniques have been developed to create genetic variants with desirable traits,including ethyl methane sulfonate(EMS)treatment,gamma irradiation,fast neutron bombardment,and T-DNA insertion.But all techniques are dependent on chance and do not produce sequence-specific mutations[5].

The rapid pace of innovation in molecular genetics is bringing revolutionary changes to conventional genome manipulation strategies.A historic breakthrough year was 1978,when Ti plasmids were first identified as being involved in DNA transfer to plants[6].In 1987,CRISPRsequences were found in a bacterium(Escherichia coli)[7].Genome editing was put on a fast track starting in 1993,when the first transgenic tomato was commercialized in USA.Since then,genome-editing with predetermined and precise changes to genomes has revolutionized crop breeding.Identification and characterization of novel nucleases in genome editing has increased the range ofprecise site-directed modifications.Engineered nucleases are used to create doublestranded breaks(DSBs)at a specific site in the genome,thus inducing a desired DNA modification at or near the break site upon repair with errors.Over the last decade,different nucleases capable of generating targeted DSBs have proven their editing efficacy in a wide range of plant species.

The past few years have witnessed the rapid adoption of genome-editing systems in the genetic improvement of polyploid crops.To date,genome-editing techniques have been used in sugarcane[9],citrus[10],grapes[11],Camelina sativa[12],wheat[13-16],tobacco[17-20],potato[21-23],flax[24],rapeseed[25-30],dandelion[31],Lotus japonicus[32],soybean[33-37],and cotton[39].Given the robustness of genome-editing systems in generating site-specific DNA mutations,the conventional gene manipulation approaches are being replaced with site-specific nucleases(naturally occurring restriction enzymes that have extended DNA recognition sequences)mediated genome editing systems.In this review,we describe several genome-editing systems and their applications in genetic improvement of polyploid crops.

2.Genome-editing techniques

Four main kinds of site-specific nucleases(SSNs)that have been engineered for plant genome editing,include meganucleases(MNs),zinc-finger nucleases(ZFNs),transcription activator-like effector nucleases(TALENs),and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9).

2.1.Meganucleases

MNs or homing endonucleases(HEs)are highly specific endonucleases with large cleavage sites(＞14-40 bp,Fig.1-A)that can induce homologous recombination in cultured cells,including mammalian cells and plants.They are the first reported SSNs used to produce targeted DSBs at specific loci in eukaryotic genomes.The DSBs produced by MNs can be repaired by nonhomologous end joining(NHEJ)or homology-directed repair(HDR)[38].The engineering of MNs is challenging,because their DNA recognition and cleavage functions are intertwined in a single protein domain(Fig.1-A)[8,40].On the basis of sequence and structural motifs,homing endonucleases are divided into five families:GIY-YIG,HNH,His-Cys box,PD-(D/E)XK,and LAGLIDADG[41].Homing endonucleases can tolerate target-site polymorphism without loss of binding and cleavage activity.A long exact target site occurs rarely in the genome.For example,only one I-SceI targetsite is found in the 13 Mb yeast genome[40].Meganucleases technology has worked well with I-SceI-mediated recombination in mice,bacteria,mosquitoes,plants,and flies.This technology serves as a genome-editing tool(Tables 1)that allows targeting of chromosomal locus in various genomes[42-46].But owing to the target-sequence specificity of meganucleases,the technology is not commonly used.

Fig.1-(A)Meganucleases(MN)or homing endonucleases are considered mobile introns,and their small size and large DNA recognition site make them an attractive option for genome editing.(B)Zinc finger nucleases(ZFNs)consists of a DNA-binding(Cys2His2)and a DNA cleavage domain(Fok I restriction enzyme)which are designed to bind DNA in tail to-tail orientation.Dimers create DSBs that can induce mutations at the target site.The Fok I domain has characteristics that support targeted cleavage.(C)Transcription activator-like effector nucleases(TALENs)consist of DNA binding(TALE DNA)and cleavage domains.The TALE DNA binding domain consists of repeats with amino acids that facilitate binding to a single DNA base.Monomers are normally designed with 15-20 RVDs and the target site is typically＞300 bp.(D)Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)consists of Cas9 endonuclease and a gRNA.sgRNA forms a complex with the Cas9 nuclease.The target site is chosen from a targeted DNA sequence directly upstream PAM site(NGG).

2.2.Zinc-finger nucleases(ZFN)

ZFN which are chimeric fusion proteins,consist of a DNA-binding domain with a DNA recognition module and a DNA-cleavage domain(Fig.1-B).DNA-binding domain is composed of a set of Cys2-His2 zinc fingers,each recognizing 3 to 6 bp of DNA.DNA-cleavage domain or array of engineered zinc finger proteins(ZFPs)fuses with the catalytic domain of the Fok I restriction enzyme.Dimers of ZFNs can create DSBs in DNA of living cells,which can induce highly efficiency mutation on the target site[47-49].Fok I domain is the backbone of ZFN because it has many characteristics that support targeted cleavage in a complex genome[50].The major constraint of ZFN-mediated genome modification is its time-consumingnature,low efficiency,and low reproducibility in germ cells though typically high efficiency in somatic cells[51].Polyploid crops successfully edited with ZFN include tobacco[17]and rapeseed[25](Tables 1 and 2).

Table 1-Genome-editing technology used against biotic stresses.

2.3.Transcription activator-like effector nucleases(TALENs)

The plant bacterial pathogen Xanthomonas infects many crop species including rice,citrus,tomato,and soybean[52].A battery of proteins delivered by Xanthomonas to plants is called transcriptional activator-like effectors(TALEs).One of the primary experiments was to fuse the TALE DNA binding domain with the catalytic domain of the Fok I endonuclease,resulting in TALENs.The large target site makes TALENs more specific than other nucleases because TALEN monomers are normally designed with 15-20 repeat variable residues(RVDs)and a TALEN target site is typically＞30 bp(Fig.1-C)[53].TALENs have been used in many organisms to generate targeted modification(Tables 1,2,and 3).The advantage of TALENs is that they allow targeting on average one DNA locus every 10 bp,an advantage over ZFNs.DNAs binding domains of TALENs can easily be engineered to recognize virtually any DNA sequence[54,55].In both ZFNs and TALENs,there is a major hurdle to widening their applications:a new chimeric protein must be engineered for each target sequence of interest.Engineering a new protein for each new target is a complex and time-consuming process that it is not feasible in all laboratories.TALENs have a very large size,from～950 to～1900 aa per pair,so that delivery of TALENs to plant cells is also challenging.In addition to TALENs,a new de novo engineered transcription activator-like effector(dTALEs)that helped to activate or suppress gene expression in plants has been developed[56,57].

2.4.CRISPR/Cas9

The most recent addition to the genome-editing toolbox,that of the clustered regularly interspaced short palindromic repeats(CRISPR)system has revolutionized the genome editing system.Beyond enabling facile and efficient targeted genome editing,it has the potential to regulate endogenous gene expression or to label specific chromosomal loci in living organisms or cells[58].The simplicity and robustness of this system,has made it an easy to use and attractive genome-editing tool worldwide.It consists of a chimeric RNA and a single monomeric protein,Cas9(Fig.1-D).Cas9 consists of two lobes including a large globular recognition(REC)lobe and a small nuclease(NUC)lobe[59].The single guide RNA(sgRNA)is the second component of the CRISPR/Cas9 system and forms a complex with the Cas9 nuclease.In CRISPR/Cas9 system,5′-NGG or 5′-NAG is recognized as protospacer adjacent motif(PAM)site.Upstream of PAM site,18-22 bp sequence is selected as target region.

Table 2-Genome editing to enhance quantity,quality,and shelf life.

Table 3-Important traits of crops developed by genome editing.

3.Application of genome-editing techniques in polyploid crops

The main objective of genetic improvement of crops is to increase food production.Polyploid crops comprise a large proportion of major food,fiber and industrial crops include rapeseed,wheat,potato,cotton,apple,peanut,sugarcane,and citrus.In most polyploids,their genome complexity poses a challenge to generate genetic mutation by conventional biotechnological strategies[60].However,the rapid adoption and application of SSN-mediated genome-editing system have greatly accelerated the process in crop genetic improvement.

3.1.Bacterial resistance

Bacteria can cause diseases in plants and produce many metabolites including toxins,polysaccharides,pectic enzymes,and hormones.Pectic enzymes degrade pectin layers in plant cells and generate crown gall tumors[61].When citrus CsLOB1 genes were targeted using the CRISPR/Cas9 technique[10],edited plants showed high resistance against the bacterium Xanthomonas axonopodis.In addition,deletion of the entire effector-binding elements(EBEPthA4)sequence from both CsLOB1 alleles conferred a high degree of bacterial resistance in homozygous mutants generated from citrus explants.

3.2.Fungal resistance

Modern agriculture is dependent on chemicals to avoid losses caused by fungal disease.The application of chemicals in intensive farming impairs human health and the environment.Development of fungus-resistant wheat cultivars has been a major objective of wheat breeding.Genome editing precisely knocked out genes that confer susceptibility to fungal pathogen in wheat[13].Simultaneous targeting of the TaMLO-A1,TaMLO-B1,TaMLO-D1 genes with the CRISPR/Cas9 and TALEN technologies using particle bombardment,resulted in powdery mildew resistance.Similarly,Fusarium head blight(FHB)resistance was also achieved in wheat by targeting three wheat genes including an ABC transporter(TaABCC6),the nuclear transcription factor X box-bindinglike1(TaNFXL1)and a gene encoding a nonspecific lipidtransfer protein(nsLTP),TansLTP9.4[16].These three genes were edited with 42.2%mutation efficiency and were found to be involved in defense against FHB.

3.3.Insect resistance

The application of insecticides in agriculture damages and destabilizes the ecosystem.Agriculture is moving towards biological control of insects,including the use of crop cultivars with insect resistance.Polyploid cotton has a complex genome which is very hard to exploit due to incomplete genome sequence information. A re-engineered meganuclease was used for specific cleavage of an endogenous target sequence adjacent to a transgenic insect control locus in cotton[39].The combination of targeted DNA cleavage and homologous recombination repair made possible the insertion of additional genes.Mutation in flanking regions of the cry2Aelbar gene and repair with the herbicide tolerance genes including 5-enolpyruvylshikimate-3-phosphate(EPSPS)and p-hydroxy-phenyl-pyruvate dioxygenase(HPPD)resulted in stacking of herbicide-tolerance and insect-resistance stacking.

3.4.Virus resistance

Viruses may damage all plant parts,including flowers,seeds,roots,stems,and leaves.As strict intracellular pathogens,viruses are hard to control chemically.Prophylactic measures damage infected plants and excessive pesticide application is needed to limit virus carrier populations.Conventional breeding has produced few examples of incorporation of complete virus resistance into a cultivar.Efficient and durable resistance that can withstand the extreme genetic plasticity of viruses in polyploid crops is needed.In an attempt to develop virus-resistant Nicotiana benthamiana plants,an sgRNAs were designed for specifically targeting the coding and non-coding sequences of tomato yellow leaf curl virus(TYLCV).CRISPR/Cas9 targeted TYLCV by inducing mutations at the target region,resulted in transgenic N.benthamiana plants with delayed or reduced accumulation of viral DNA,with no or less symptoms of viral infection[62].

3.5.Herbicide tolerance and resistance

Farmers try to remove unwanted plants from fields by tillage,hand weeding,and herbicide application.Tillage practices cause many problems by exposing the top layer of soil to wind and water erosion,as well as increasing labor inputs.Development of herbicide-resistant crops is an efficient approach to weed control in modern agriculture.In the first cases of genome editing,the acetolactate synthase genes(SuRA and SuRB)were targeted in tobacco plants to achieve resistance against imidazolinone and sulfonylurea,using ZFN technique with electroporation of protoplasts.Herbicide-resistance mutations were introduced successfully into SuR loci and＞40%of recombinant plants showed modifications in SuR alleles[17].In Arabidopsis thaliana,5-enolpyruvylshikimate-3-phosphate(EPSPS)gene was successfully edited using single-stranded oligonucleotides(ssODN),TALEN,and CRISPR/Cas9.High targeted genome-editing frequency was achieved when ssODN was simultaneously delivered with TALEN or CRISPR/Cas9 component[24].After successful editing of EPSPS genes in a model plant,ssODN and CRISPR/Cas9 were delivered to flax plants.Edited flax plants showed high tolerance to glyphosate compared with control plants.In potato,a geminivirus replicon was used to transform CRISPR/Cas9 components at the target site of the acetolactate synthase 1(ALS1)gene and mutated transgenic free plants showed reduced susceptibility to herbicides[21].

3.6.Enhanced quality

For genetic modification of quality traits,the L-idonate dehydrogenase gene(IdnDH)in polyploid Vitis vinifera was targeted with CRISPR/Cas9 to control tartaric acid,which determines color and taste as well as yeast growth during fermentation[11].TALEN technology was used in potato to target the vacuolar invertase(Vinv)gene,generating a reduced level of acrylamide to increase quality and remove carcinogenicity[22].Soybean oil is high in linoleic acid,a polyunsaturated fatty acid that is undesirable for cardiovascular and cerebrovascular health.The fatty acid desaturase 2(FAD2-1A and FAD2-1B)genes,which encode enzymes catalyzing oleic acid desaturation,were targeted successfully in soybean,resulting in a drastically changed fatty acid profile.The content of oleic acid was increased from 20%to 80%and that of linoleic acid was decreased from 50%to 4%after mutagenesis in soybean[34,35].In a similarstudy,FAD2 genes were targeted with CRISPR/Cas9 to produce mutagenesis in polyploid rapeseed.After a FAD2 site(BnaA.FAD2.a)was targeted,mutant plants with the fad2-Aa allele were selected from backcross(BC1)progenies and homozygous plants were produced in self(BC1S1)progenies.This metabolic pathway change increased the oleic acid content in mutant plants relative to wild-type plants[28].To improve fatty acid quality,the B.napus β-ketoacyl-ACP synthase II(KASII)gene was successfully targeted with ZFN technology in another experiment[25].Transgenic progenies expressing transcriptional activators(ZFP-TFs)were shown to have elevated KASII transcript levels and decreased palmitic acid and increased total C18 acids.Camelina sativa is a flowering plant also known as gold of pleasure or false flax.The application of CRISPR/Cas9 technology to target the CsFAD2 gene,reduced the level of polyunsaturated fatty acids and increased that of oleic acid[12].Potato granular-bound starch synthase(GBSS)genes were altered with CRISPR/Cas9 technology for amylose synthesis and the ratio of amylopectin to amylose was increased by effective multi-allelic mutagenesis[23].

3.7.Increased yield

Many genes influencing yield-component traits have been modified,and include those for number of kernels per panicle/pod/bear,kernel weight,and number of panicles per plant.To generate high-yielding plants by genome editing,the wheat TaGASR7-associated with grain length(member of gibberellic acid stimulated Arabidopsis GASA-gene family)and dense-erect panicle(TaDEP1)genes were targeted with CRISPR/Cas9 and mutant plants were generated with mutations in six alleles.Mutation in all six alleles of TaGASR7 increased thousand-kernel weight[14].In a similar study,multiplexed genome editing with CRISPR/Cas9 was performed to target the wheat grain weight(TaGW2)gene.Knockout mutants showed increased seed size and thousand-kernel weight[15].To produce more branches and etiolated leaves,the N.benthamiana genome was targeted with CRISPR/Cas9 at phytoene desaturase(NtPDS)gene and NtPDR6 loci(pleiotropic drug resistance transporters and homolog of Petunia hybrida,PDR1),resulting in etiolated leaves in PDS mutants and more branches in PDR6 mutants[18].In tetraploid B.napus,the CLAVATA-3(CLV3)gene involved in secreting peptides and its associated receptor genes including CLV1 and CLV2 were targeted with the CRISPR/Cas9 system[30].Double mutation of BnCLV3 produced more leaves and multilocular siliques in mutant plants.The multilocular siliques showed increased numbers of seeds per silique and higher seed weight than the wild type[30].

3.8.Other traits improved by genome editing in polyploid crops

Genome editing opens a new avenue to mutagenesis by insertion,deletion,and gene replacement that has been adopted in many other polyploid crops.In particular,CRISPR/Cas9 was successfully used to target the Glyma07g14530,Glyma06g14180,Glyma08g02290,and Glyma12g37050 genes of soybean to transform hairy roots in mutants and nodules in tissues[36,37].Glycoproteins were produced in N.benthamiana mutants with a TALEN-based strategy by targeting the fucosyl transferase(Fuct)and xylosyl transferase(XyIT)genes[19].The XyIT and FucT genes were also mutated with CRISPR/Cas9 to knock out their function for the complete inactivation of β(1,2)-xylose and α(1,3)-fucose production in N.tabacum[63].To increase the production of bioethanol,an emerging fuel worldwide with the potential to reduce globalwarming and pollution,the sugarcane genome was targeted using a TALEN strategy to manipulate caffeic acid O-methyltransferase(COMT)to improve cell wall composition[9].Targeting the COMT locus site led to a large increase in bioethanol production.In dandelion,CRISPR/Cas9 system was used to mutate for the producing high molecular-weight rubber in roots[31].The Fructan:Fructan 1-fructosyltransferase(1-FFT)gene was targeted for increasing rubber production.The mutation rate varied from 15.2%to 80.0%in regenerated plants.All types of mutations were detected,from one to 12 nucleotides,resulting in amino acid deletions,frame shifts,and introduction ofpremature stop codons.In B.napus,the ALCATRAZ(ALC)gene was mutated with CRISPR/Cas9[26].Itwas found thatonly 5-6 cmlong siliques of mutated plants were resistant to pod shatter whereas all shorter siliques showed no differences in pod shatter compared to the wild type.This finding may shed light on the genetic improvement of shattering resistance in rapeseed.In N.tabacum,the plant architecture-controlling genes carotenoid-cleavagedioxygenase-8A and-8B(NtCCD8A,NtCCD8B)were targeted with CRISPR/Cas9.In the mutated plants,plant height and total biomass were reduced,while numbers of branches and leaves were increased,with the root-to-shoot ratio unchanged[20].

4.Conclusions and perspectives

In plant breeding and genetics,innovations are essential to meet the challenges of world masses.Crop improvement requires the continuous creation and deployment of new allelic variants.Genome-editing technology,especially the CRISPR/Cas9 system,provides a valuable platform for generating mutants with high frequency in polyploid crops.Genome editing gives hope for improving crops to achieve future food security.As mentioned above,it is expected to improve agronomic traits including crop yield,quality,appearance,shelf life,and stress resistance.Moreover,these techniques have great potential to facilitate whole-genome functional studies leading to applications in polyploid crops.

4.1.The CRISPR/Cas9 system facilitates the rapid application of genome editing technology in polyploid crops

Although SSNs for constructing genome editing systems have been described,different SSN-mediated genome-editing systems have their own characteristics.Both ZFN and TALEN are used to mutagenize a genome at a specific locus.These systems require two different DNA binding proteins flanking a sequence of interest,each with a C-terminus Fok I nuclease module.Because these methods have not been widely adopted,few crops have been edited with ZFN and TALEN.MNs are also not commonly used in plants.But they have served as a launching pad on which the scientific community has been able to develop new techniques.The CRISPR/Cas based genome system has emerged as an effective strategy for crop genetic improvement.By using gRNA,it can precisely induce DNA mutation at a designated genome site.Multiplexed genome editing is becoming a preferred approach in polyploid crops.By this approach,genome-editing can be achieved by expression of gene constructs having multiple single gRNA-expression cassettes.

As a case study for multiplexed genome editing,in polyploid B.napus,12 genes including four paralogous genes of the RGA family involved in the repression of gibberellic acid,three paralog FUL genes involved in regulating pod dehiscence and five paralogous genes of the DA2 and DA1 families contributed in negative regulation of organ size,were targeted.In the T0 generation,a 65.3%mutation frequency was observed.But for paralogous genes located in conserved regions,mutation frequencies of 27.6%-96.6%were observed.Homozygosity,biallelism,and heterozygosity were stably inherited in Mendelian fashion in the T1generation with an overall genetic mutation rate of 48.2%.No putative off-targets was observed,indicating high mutagenic efficiency and specificity of CRISPR/Cas9 in polyploid rapeseed[29].In a similar study,an efficient CRISPR/Cas9 platform was developed in polyploid B.napus to target five homologous of the SPL3 gene[27].The editing frequency of different homoeologous gene by CRISPR/Cas9-induced mutagenesis was observed ranging from 96.8%to 100.0%.

4.2.Strategies for applying CRISPR/Cas9 system in polyploid crops

To date,although CRISPR/Cas9-mediated genome editing has been extensively exploited in plants,its editing efficiency has varied dramatically.Especially in polyploid crops,the presence of paralogs and orthologs with functional redundancy requires the difficult simultaneously knockout of all copies of genes with the same function.Optimization of Cas9 codon,promoters and target sequence composition(GC content)may directly affect mutagenic efficiency in polyploid crops[64-68].Designing sgRNA design is more difficult in polyploid crops than in diploid species such as Arabidopsis and rice.Although some CRISPR/Cas9 tools(CRISPR-P and CRISPR-P2.0)for sgRNA design have been developed,they still have limitations in polyploid crops[69,70].To knock out homologous genes(paralogs and orthologs)simultaneously,it is necessary to design sgRNA from a conserved region that can target all gene copies.To target all gene copies or a specific gene copy,sgRNA can be manually designed after sequence analysis.If no conserved region is found for a large number of homoeologous genes,it is necessary to divide the genes into more than one group and design sgRNAs based on a conserved region in each group.

New orthologs of Streptococcus pyogenes SpCas9 have been shown effective for plant genome editing including S.thermophilus(stCas9)and Staphylococcus aureus(SaCas9)[71].The size of the commonly used RNA guided endonuclease Cas9 from S.pyogenes limits its use in genome targeting.SaCas9 from Staphylococcus aureus also targets the genome with high efficiency similar to that of SpCas9,though＞1 kb shorter in length than SpCas9,and thus easily be delivered for genome targeting[72].CRISPR/Cpf1,a new class of CRISPR system,was recently demonstrated to be as effective as CRISPR/Cas9 for plant genome editing[73].The PAM of CRISPR/Cpf1 is TTTN,which is suitable for targeting an AT-rich genome sequence,such as in promoter regions of plant genomes.CRISPR/Cpf1 generates DSB with 5′sticky ends,facilitating gene mutation by NHEJ during repair.Recently,several expanded PAM of Cas9 and Cpf1 variants have been identified,broadening the recognition site of PAM sequences.These new variants will further facilitate sgRNA design in genome editing of polyploid crops.

CRISPR/Cas-mediated precise base editing is another important application for crop improvement.By introducing a base-editing function into CRISPR/Cas9,cytidine deaminase directly substitutes cytidine with uridine by inducing C→T and G→A conversion[74,75].A base editing system in wheat[78]has the potential to be used for site-specific modification in other polyploid genomes.

4.3.New approaches for overcoming concerns regarding genetically modified organisms(GMOs)and off-target effects

To obtain transgenic plants,several approaches have been developed for delivering CRISPR/Cas9 components into plant cells,including Agrobacterium tumefaciens-mediated and biolistic delivery.An efficient A.tumefaciens-mediated approach has been established in many polyploid species such as rapeseed[27]and cotton[76].Although this approach represents a simple and efficient method for transforming CRISPR/Cas9 reagents into plant genomes,it unavoidably introduces foreign DNA fragments,increasing the chance of off-target mutation and genome contamination.

An approach using three high-fidelity SpCas9 variants,including eSpCas9(1.0),eSpCas9(1.1),and SpCas9-HF1,has been developed to reduce off-target mutations in CRISPR/Cas9-mediated genome editing[77].All three SpCas9 variants showed a requirement of precise 20-nt guide sequence derived from tRNA-sgRNA precursors for efficient and precise on-target editing in rice protoplasts.Even one extra nucleotide at the 5′end of the guide sequence increases the chance of off-target.In polyploid crops,off-target effects may also be avoided by use of a 20-nt guide RNA for precise genome editing.

Recently,a DNA-free genome editing system involving the delivery of preassembled ribonucleoprotein complexes(RNPs)or CRISPR/Cas9 in vitro transcripts(IVTs)into plant cells by biolistic transformation in allohexaploid bread wheat has been described[78].Given that RNPs and IVTs would be rapidly degraded in plant cells,such approaches will reduce off-target effects and generate transgene-free mutations in plant genomes.This approach will reduce public concern about biosafety.Public acceptance will be needed for the application of genome editing technology in all crops.

Acknowledgments

This work was supported by the National Natural Science Foundation of China(No.31700316),the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences(Group No.118),the Earmarked Fund for China Agriculture Research System(CARS-12),the Fundamental Research Funds for Central Non-Profit Scientific Institution(1610172018009),and Graduate School of Chinese Academy of Agricultural Sciences.