Wheat genome editing expedited by efficient transformation techniques: Progress and perspectives

*

Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081,China

1.Introduction

The challenge of feeding a global population of 9 billion by the middle of this century is enormous[1].Common wheat as a staple food crop will play a major role in meeting this challenge.Wheat has lagged behind other major cereal crops in development of genetic engineering and biotechnology because of its huge genome,high number of repetitive DNA sequences,hexaploid composition,and low regeneration following genetic transformation[2].No transgenic wheat has been commercialized and new wheat varieties are mainly developed by conventional breeding techniques that are costly and time-consuming[3].The key reasons for this is lack of a high quality reference genome sequence and difficulty in transforming wheat.

Plant transformation with exotic genes using vectors like Agrobacterium has been the first step in introducing genes of interest to plant cells that must be regenerated into plants that produce normal seeds.Common wheat as a hexaploid plant is one of the most difficult crops to be transformed.Recently,a new technique called Pure Wheat that significantly improves transformation efficiency was invented by the Japan Tobacco Company[4].This technique brings the hope of genetically manipulating wheat in a more efficient and diverse manner.It also enables application of new genome editing technologies that are applicable to wheat.

Genome editing as a recently developed technology enables precise manipulation of specific genomic sequences,and will possibly supersede traditional random mutagenesis methods in plant breeding. In general genome editing technologies involve three types of sequence-specific nucleases(SSNs),namely zinc-finger nucleases(ZFNs),transcription activator-like effector nucleases(TALENs),and clustered regularly interspaced short palindromic repeat-associated endonucleases(CRISPR/Cas).Such technologies have versatile functions including targeted gene knock-out and knock-in,gene replacement and activation,and DNA repair[5–8],and will be widely applied in crop breeding.This is likely to be led by the application of CRISPR/Cas9 with the help of plant regeneration-related genes such as Baby boom(Bbm)and Wuschel(Wus2)in co-transformation[9].

In this review,we provide a brief summary of current transformation techniques and recent breakthroughs in genetic engineering of wheat.We then review recent progress in plant genome editing and its application in wheat.Finally,we speculate future trends in wheat genetic engineering with the availability of a high quality genome sequence, a significantly improved transformation protocol,and a tool for genome editing in generating elite wheat varieties that will contribute to achievement of world food security.

2.Wheat transformation-possible after much effort

2.1.Transformation of wheat by biolistic particles

The first transgenic wheat plants were obtained by biolistic particle bombardment in 1992[10].The Bar gene as a selective marker was successfully transferred to wheat by high velocity microprojectile bombardment.This was the beginning of the era of wheat transformation.A number of genes were transformed into wheat using this approach(Table1),including functional genes such as TaPIMP1[20],Yr10[22],TcLr19PR1[23],TaNAC2[26],and TaCPK[27].Genetically enhanced wheat lines that have better resistance to biotic and abiotic stresses are still being tested.However,use of biolistic particles is notorious for its low transformation efficiency.

2.2.Wheat transformation by Agrobacterium

Agrobacterium species used for wheat transformation include A.tumefaciens and A.rhizogenes.They use a transfer DNA(T-DNA)that naturally integrates into plant genomes after infection.Agrobacterium mediated transformation has specific advantages compared to biolistic particles,including low copy number integration,a more economic and simpler procedure,and clear integration of sequences without the vector backbone.Although it is widely used by wheat scientists(Table 2),the efficiency of Agrobacterium mediated wheat transformation using immature embryos remained extremely low until recently,when the PureWheat technique was developed by the Japan Tobacco Company.This revolutionary technique involved cultivar Fielder as a host with various modifications in transformation protocols.The efficiency of this method has reached as high as 50%[4].The technique was confirmed in Australia using wheat cultivars Westonia and Gladius[40].With additional modifications the present authors'laboratory has transformed more than 15 Chinese genotypes,including elite varieties Jimai 22,Shiluan 02-1,Yangmai 16,Jimai 5265,Zhoumai 18,and Lunxuan 987,with high Agrobacterium infection efficiency and less genotype dependence(Fig.1)[39].

From our experience and that of others,a number of factors need to be considered in order to achieve high transformation efficiency.Firstly,the infection efficiency should be high.Wheat genotypes differ in susceptibility to Agrobacterium infection.Secondly,the wheat genotype should have high regeneration ability,as exemplified by Bobwhite,Fielder,Kenong 199,and Yangmai 158[4,26,31,41].Thirdly,the plants from which immature embryos are collected should bein good growth status.High temperatures in particular have adverse effect on transformation success rates[42,43].Based on our experience,mild temperatures,strong light,and good cultivation management are beneficial for high transformation efficiency[39].Transformation efficiency can be increased using the morphogenic regulator genes BABY BOOM(Bbm)and WUSCHEL2(Wus2)that have been successfully tested in maize[9].However,over-expression of Wus2 may lead to callus necrosis as well as aberrant callus phenotypes.The morphogenic regulator genes have to be excised during regeneration.

Table 1––Examples of wheat transformation achieved by biolistic particles.

3.Development of genome editing systems in wheat

3.1.The advance of plant genome editing technologies

In the past few years genome editing technologies have arisen as promising tools for crop improvement,including wheat.The earlier versions are represented by the CRISPR/Cas9 system that largely replaces the older versions such as ZFNs and TALENs due to its simplicity and economy(Table 3).The CRISPR/Cas9 system has two simple components;the Cas9 protein and the single guide RNA(sgRNA)that guides the nuclease Cas9 to target sites in a sequence-specific manner where DNA double-strand breaks(DSBs)are generated.DSBs are repaired by one of the two main competing DNA repair pathways:error-prone non-homologous end joining(NHEJ)that results in small random insertions and/or deletions(indels)at the cleavage site,or homologous recombination(HR)that leads to precise genome modification[5–8].NHEJ can be utilized to generate random changes and mutations in target sites where HR can carry out targeted knock-in,gene replacement,and DNA correction.The design of multiple sgRNAs to target multiple sequences allows multiplex high-efficiency genome engineering[44,45].These applications are extremely useful in genetic manipulation for crop improvement.

In addition to conventional functions of genome editing,such as gene insertion,gene replacement and regulation of gene expression[46–51],a catalytically inactive or ‘dead'Cas9(dCas9)that bears mutations in both the RuvC D10A and HNH H840A domains and is nonfunctional as a nuclease can be combined with proteins having other functions[52].For example,transcriptional activation or repression domains can be fused with the dCas9 to regulate endogenous gene expression[53].Additional examples are fusion of dCas9 withchromatin-modifying enzymes such as inhibitors of histone deacetylases or DNA methyltransferases to achieve altered epigenomes and transcriptomes[54–58].Other examples include light-sensitive proteins fused with dCas9 that allows rapid and reversible targeted gene activation or repression by light[59–61].

Table 2––Examples of wheat transformation mediated by Agrobacterium tumefaciens.

3.2.New versions of CRISPR/Cas9 systems

Since the discovery of the CRISPR/Cas9 system,a number of different versions have followed.Unlike Cas9 that is derived from Streptococcus pyogenes(SpCas9)and recognizes a relatively simple PAM(5′-NGG-3′),a new Cas9 protein from Neisseria meningitides(NmCas9)recognizes a more complex PAM(5′-NNNNGATT-3′)[62].The longer PAM reduces the accessible target range and hence potential off-target sites,leading to more precise genome editing[63].Cas9 proteins derived from other microorganisms such as Staphylococcus aureus(SaCas9)and Campylobacter jejuni(CjCas9)also have different PAMs,i.e.,5′-NNGRRT-3′and5′-NNNNRYAC-3′,respectively[64,65].CjCas9 is 984 aa in length and is the smallest Cas9 protein identified so far[65],which makes the development of expression cassettes for delivery into plant cells much easier.

Besides Cas9 proteins,other nucleases with similar genome editing functions are reported,such as Cpf1,C2c1,and C2c2 proteins[66,67].Cpf1,for example,has a RuvC-like nuclease domain,but does not have any of the HNH nuclease domains that are commonly present in Cas9.Cpf1 is a single RNA-guided endonuclease lacking tracrRNA,and recognizes a T-rich PAM motif.In addition,it not only cleaves target DNA but also processes its own CRISPR RNA(crRNA)[66,68].Moreover,itgenerates staggered ends with four-or five-nucleotide overhangs,which are useful for increasing the insertion efficiency of a desired DNA fragment into the cleaved site using complementary DNA ends during HR.Application of Cpf1 might significantly enhance gene insertion efficiency at a precise genome location,a highly desirable attribute.The CRISPR-Cpf1 system has been widely tested in animals[67]as well as plants such as rice,Arabidopsis,soybean,and tobacco[69,70].

The second Cas9-likeprotein isC2c1,which isa dual-RNA-guided DNA endonuclease that generates a staggered break of target DNA with a six-to-eight nucleotide(nt)overhang at 5′ends.This endonuclease is reported to be highly sensitive to nucleotide mismatches to the target DNA[71].C2c2 is the latest developed technique that enables genome editing at the RNA level[72].C2c2 is an RNA-guided RNA-targeting effector.It contains two highly conserved R-X-H motifs that comprise the typical higher eukaryote and prokaryote nucleotide-binding HEPN domain,but lacks an identifiable DNase catalytic site[73].The RNA molecule in this system is cleaved outside of the base-paired region by the C2c2 complex.In addition,the C2c2 complex can also cleave other RNA molecules in trans in a sequence-nonspecific manner[74].

3.3.Single-base editing—an ultra-precise technology

Fig.1–GUS transient expression in immature embryos of various Chinese wheat varieties after five days of co-cultivation with Agrobacterium.

A recently invented single base editing approach enables a direct,irreversible conversion of a single-nucleotide base to another base in a programmable manner[75].This method does not generate DSB cleavages or require a donor template,and does not induce an excess of stochastic insertions and deletions.In principle,dCas9 is fused to a cytidine deaminase enzyme that mediates the direct conversion of cytidine to uridine,thereby leading to the change of a C/G pair to a T/A pair.Base editing would convert cytidines within a range of approximately five nucleotides.Four kinds of cytidine deaminase enzymes(human AID,human APOBEC3G,rat APOBEC1,and lamprey CDA1)were detected for base editing efficiency and rat APOBEC1 showed the highest deaminase activity in human cells[75].Target-AID (target-activation induced cytidine deaminase)comprised of dCas9 fused to Petromyzon marinus cytidine deaminase(PmCDA1)has been used to generate point mutations in tomato(Solanum lycopersicum)[76]and rice[77].

3.4.Genome editing in wheat

Chinese scientists are leading the genome editing effort in wheat.The first successful case of using the CRISPR/Cas9 system was in wheat protoplasts where mutations of wheatdisease resistance gene TaMLO were generated with a mutagenesis frequency of 28.5%[78].Later,fertile transgenic wheat plants with edited genomes were produced by the older genome editing method TALENs[79].The disruption of all three TaMLO homoeologs generated plants with resistance to powdery mildew.Soon thereafter,transgenic wheat plants carrying mutations in the TaMLO-A1 allele were generated by Cas9 technology.Moreover,the GFP,His-tag and Myc-tag genes were successfully inserted into desired loci in wheat protoplasts.A detailed CRISPR/Cas-mediated mutagenesis protocol in wheat protoplasts was recently described for target sequence selection,construction,and verification of sequence-specific sgRNAs[80].

Table 3–Comparisons of ZFNs,TALENs,and CRISPR on genome editing.

Targeted genes TaGASR7,TaGW2,and TaLOX2 in hexaploid heat and TdGASR7 in tetraploid durum wheat that are associated with grain yield or disease resistance,were successfully edited by transient expression systems of TECCDNA and TECCRNA;in particular,homozygous mutants and transgene-free plants were obtained in the T0generation[81].Moreover,by delivering CRISPR/Cas9 ribonucleoproteins into immature wheat embryo cells,editing of genes TaGW2 and TaGASR7 was achieved in varieties Kenong 199 and YZ814 without transgene integration[82].A dCas9 fused with cytidine deaminase enzyme enabled direct and irreversible programmed conversion of one target DNA base into another,i.e.,‘base editing'was also reported in wheat[83].In this case,the coding sequence of blue fluorescent protein(BFP)was edited to produce green fluorescent protein(GFP)by changing the 66th codon CAC(histidine)to TAC(tyrosine)without double-strand breaks or application of a foreign DNA donor.A similar single base edition,a C to T substitution,was made for the TaLOX2 gene[83].We expect the leap in wheat transformation efficiency will lead to a functional genomics research era in wheat.

4. Perspectives in developing genetically engineered wheat

4.1.Increased transformation efficiency provides a jump start for genome editing

Until recently it was difficult to imagine that wheat transformation efficiency could reach 50%in any genotype[4].The realization of highly efficient transformation and improvement in techniques that remove the obstacle of genotypic limitation provides a launching pad for functional studies and application of new technologies such as genome editing.Transformation will soon no longer be a bottleneck for genome editing in wheat.Despite this,further efforts are needed to progress genetic engineering to a level of developing elite varieties that meet world food requirements.

Overcoming genotype dependence is a revolutionary breakthrough in plant transformation.This breakthrough first came in maize.Co-overexpression of morphogenic regulator genes Bbm and Wus2 by Agrobacterium-mediated transformation of immature embryos led to high transformation frequencies in several previously recalcitrant maize inbred lines[9].In addition,the combined use of Bbm and Wus2 enhanced transformation efficiency in sorghum,rice,and sugarcane.These kinds of genes can also be used in wheat to solve the bottleneck of genotype dependence in plant regeneration in further improving transformation efficiency and accelerating the application of genome editing technology.

4.2.Additional measures to further increase transformation efficiency

Most current wheat transformation protocols require immature embryos for callus production.High quality immature embryos demand well-grown plants that are mostly produced in environments that require extra inputs and costs.Use of mature embryos would remove seasonal limitations and the need to maintain high-cost conditions for plant growth.

The second measure to further increase wheat transformation efficiency is to develop and use visible selection markers.This is because detection of positive transgenic wheat plants using current molecular tools and chemical markers is still time-consuming and costly.In this regard,pigment encoded genes as visible markers can be employed for direct identification of transgenic tissues or plants.In fact,transgenic rice with purple endosperm was generated by the transference of eight anthocyanin-related genes(two regulatory genes from maize and six structural genes from Coleus)[84].Purple transgenic wheat plants have also been obtained in the authors' laboratory by transferring two anthocyanin-related genes from maize(Fig.2).We can link the anthocyanin-related genes with a selective gene such as nptII,bar,or hpt,to identify transgenic wheat plants directly by color.The application of these extra measures should further increase wheat transformation efficiency to an industrial level for future commercialization.

4.3.Developmentofmarker-freeandtransgene-free engineered wheat plants

For a long time,selective markers used in transgenic plants were antibiotic resistance genes that caused a focus of public concern.Current approaches to generate marker-free transgenic plants use FLP/FRT and Cre/lox site-specific recombination,multi-auto-transformation,co-transformation,and marker-free binary vectors [85]. Among these,co-transformation is the most efficient and simple technique[86],where a co-transformed T-DNA vector that carries the selective marker can be separated from the one carrying the target gene by genetic segregation.Visible markers are an alternate way for marker-free transformation when they are linked with a selective marker.Transgenic plants with no color are marker free.The use of a visible marker linked with the genome editing cassette can accelerate detection rates for positively edited cells or plants because the presence of the color suggests expression of genome editing machinery and hence the plants have a higher possibility of being edited.

Fig.2–Primary transformation of visible markers(two anthocyanin-related genes from maize)into wheat by Agrobacterium-mediation.(a)Immature wheat embryos infected by the Agrobacterium containing a normal vector without a visible markers.(b)Expression of visible marker genes in immature wheat embryos following Agrobacterium infection.(c)Transgenic wheat plants transformed with normal vectors without visible markers.(d)Transformed wheat plants expressing a visible marker.

The presence of genome editing technology makes transgene-free genetic engineering possible.Currently,there are three promising methods to create transgene-free plants:TECCDNA,TECCRNA,and direct transfer of TALENs or Cas9 protein into cells[81,87–89].Wheat transgene-free mutant plants without herbicide selection have been obtained by these methods[81,82].However,all three methods have a low efficiency due to lack of selection during plant regeneration from tissue culture.Transformation efficiency for the model wheat genotype Fielder was＞50%with selection whereas it was only 3%without selection pressure[40].The problem of low efficiency in generating transgene-free plants can be alleviated by use of visible markers,where transgenic cells can be selected by color after Agrobacterium infection,and transgene-free plants can be obtained in the following generation when the visible marker disappears.

4.4.Increasing the accuracy of wheat genome editing

Although many examples demonstrate that genome editing is a powerful tool for generating future elite crops,some problems remain.One problem is undesired off targeting due to the nature of the Cas9 enzyme to tolerate mismatches between the sgRNA and target DNA sites[44,90,91].A few strategies have been developed to improve the specificity of Cas9-mediated genome editing.One way to reduce off-targeting during genome editing is to use a pair of Cas9 nickases,i.e.,one Cas9 and one D10A,a nickase-mutated version of Cas9 that produces a single-strand break(SSB)[44,90].Two distinct sgRNAs can then be used,resulting in SSBs on each of the two DNA strands,leading to site-specific DSBs.The Cas9-D10A system has been tested in human cells and shown to reduce off-target activity by 50-to 1500-fold[92].Recent studies demonstrated that Cpf1 has much greater specificity than classical Cas9 nucleases[93].

For many existing elite varieties,genetic variation often occurs in a few bases[94].Current methods of identifying point mutations(such as targeted induced local lesions in genomes,or TILLING)are time-consuming and only detect limited numbers of point mutations[95].Thus,single-base editing could play a much larger role in this regard.The dCas9-cytidine deaminase system is very efficient,but is limited to the conversion of C/G to T/A,whereas C to G replacement occurs only at low frequency[77].Additional studies are needed to expand editing capabilities that should contribute to more efficient plant genetic engineering.

5.Conclusions

An efficient wheat transformation system has now been established,providing the required platform for developing genome editing technologies.It can be expected that more genes will be modified to increase yield and enhance stress tolerance. These technologies enable production of transgene-free varieties for commercialization.We can now solve food security issues by biotechnology applications.

Acknowledgments

We are grateful for financial support from the National Transgenic Key Project of the Chinese Natural Science Foundation (2016ZX08010-004,2016ZX08009001),andthe Beijing Natural Science Foundation(6162009).We thank Prof.Long Mao,Institute of Crop Science,CAAS,for critical revision and editing of the manuscript.

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