Implementations and improvements of genome editing techniques used in plant science have increased exponentially. For some crops, such as potato, the use of transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) has moved to the next step of trait development and field trials, and should soon be applied to commercial cultivation

Andersson, Mariette

Andreasson, Erik

Hofvander, Per

English

Epsilon Open Archive

Trends inGeneticsOPEN ACCESSGlossaryAgrobacterium-mediated transformation: amethod for inserting new genetic material in thegenome. The new genetic material is inserted at arandom site in the genome.Base editing (BE): a method for the conversion of abase (e.g., C→T, A→G) to induce point mutation at aspecific site in DNA. The system is based on CRISPRForumPotato trait developmentgoing fast-forward withgenome editingPer Hofvander,1Erik Andreasson,2 andin combination with a single-strand-cutting nucleaseand a deaminase.Cisgenic: an organism that has genetic elements fromits own or crossable species transferred to its genome.Clustered regularly interspaced shortpalindromic repeats (CRISPR): an immune systemfound in bacteria that has been adapted to a genomeediting technique. A CRISPR-derived RNA can beengineered to find and bind to a specific site in anorganism’s genome. This guide RNA can, togetherwith a nuclease (e.g., Cas), induce mutations at aspecific site in the genome.CRISPR associated protein 9 (Cas9): a nucleasecutting double-stranded DNA, generating DSBs.Double-strand break (DSB): a break in bothstrands of DNA. A DSB is repaired by the cell’s ownDNA repair mechanism (i.e., NHEJ or HR).Genome editing: targeted changes made in anorganism’s genome by various techniques(e.g., TALEN- and CRISPR-based methods).Glycemic index: gives a value of how quickly theMariette Andersson 1,*Implementations and improvementsof genome editing techniques usedin plant science have increased ex-ponentially. For some crops, suchas potato, the use of transcriptionactivator-like effector nucleases(TALEN) and clustered regularlyinterspaced short palindromic re-peats (CRISPR) has moved to thenext step of trait development andfield trials, and should soon be ap-plied to commercial cultivation.intake of a food causes an increase in blood glucoselevels.Homologous recombination (HR): transfer ofgeneticmaterial between two homologous strands. Ingenome editing, this process can be used by applyinga repair guide with homologous arms to the targetsite, also known as homology-directed repair.Nonhomologous end joining (NHEJ): anerror-prone DNA repair pathway after DSBs that canlead to mutations in the genome.Prime editing (PE): a CRISPR-based method usinga single-strand-cutting nuclease together with a reversetranscriptase and a PE RNA (pegRNA), with dualfunctions as both target and repair guide for the geneticedit; can be used to induce designed edits in thegenome.Ribonucleoprotein (RNP): a complex of RNA andRNA-binding protein; for genome editing, can beused by assembling a CRISPR-Cas complex in vitrofollowing transfection to cells. Hence, this DNA-freesystem ensures that no foreign DNA can be insertedin the genome, avoiding transgenesis.Single nucleotide polymorphism: a single-basevariation in a gene or an allele in a genome.Transcription activator-like effector nucleases(TALEN): a protein-based genome editing methodusing a designed protein TAL effector DNA-bindingdomain together with a nuclease to target and inducemutations at specific sites in a genome.Transgenic: an organism that has foreign geneticelements stably inserted in the genome.Transient expression: temporary expression ofgenes in a cell. The genes can, for example, encodeTALEN and CRISPR tools.Potato, an important food andstarch cropGlobal potato production has steadily in-creased over the past 30 years and it isnow one of the top-three staple foodcrops. Potato is also one of a handful ofcrops grown for the production of starch,a renewable bulk product used in variousfood and non-food applications. Cultivatedpotato is tetraploid, highly heterozygous,and vegetatively propagated. Therefore,the development of new cultivars usingtraditional breeding methods is a long-term effort. Extending the breeding toolboxwith technologies that can introduce ge-netic diversity to potato genotypes withoutinterrupting the overall genetic context isthus particularly beneficial for potato. Here,genome editing (see Glossary), especiallyTALEN- and more recently CRISPR-basedmethods, have shown the potential to be-come the next established breeding tools(Box 1). This is evident in the fact thatmost recent studies in potato include the218 Trends in Genetics, March 2022, Vol. 38, No. 3development of novel commercial traits, ofwhich we highlight a selection of in thisforum.Potato with increased tolerance tobiotic stress and herbicidesPotato is prone to many diseases, and theuse of fungicides is being questioned. Atthe same time, a number of pathogens arerapidly evolving; therefore, different breedingstrategies, particularly using genome editingtechniques (Box 2), could be a way to keeppace with this evolution from the plant side.A general approach for increased resistanceis the inactivation of so-called susceptibilitygenes. This has recently been tested byscreening seven different potato genes byAgrobacterium-mediated transforma-tion of CRISPR-CRISPR associatedprotein 9 (Cas9) and resulted in potatoplants with increased, but not full, lateblight resistance [1]. The inactivation ofgenes that were shown to give increasedresistance without apparent growth defectswere DOWNY MILDEW RESISTANCE 6(DMR6-1) and a bHLH transcription factor(CHL1). Inactivation of susceptibility genescan provide broad resistance against sev-eral diseases, has the potential to be a fairlysustainable technique, and can be com-bined with other sources of resistance. An-other approach aimed to restore truncatedproteins in a defense pathway by singlenucleotide polymorphism replacement ina caffeoyl-CoA O-methyltransferase geneusing CRISPR-Cas9. This technique hasbeen described to offer partial resistance tolate blight [2]. However, documented fielddata from all of these potato studies arecurrently lacking.Herbicide tolerance was targeted earlyin the implementation of TALEN andCRISPR in potato, where point mutationsin an ACETOLACTATE SYNTHASE (ALS)gene were induced. In addition to beinga commercially interesting trait, it is advan-tageously used during methodological de-velopments, since the trait and induceddesired mutations can be convenientlyBox 1. Genome editing for potato breeding: potentials and challengesTo date, the majority of traits added to potato using TALEN or CRISPR are based on induced double-strandbreaks (DSBs) and DSB repair by error-prone nonhomologous end joining (NHEJ). Both the stable introduc-tion of genome editing-tools in the genome, using Agrobacterium-mediated transformation, and vector and RNP-based transient application of the same tools result in a very high mutagenesis efficiency when applied to potato.Although transient application of genome editing has benefits such as low risk of obtaining chimeras and reducedrisk of induced off-targets as well as yielding transgene-free events, stable transformation is today the predominantdelivery method in research studies, plausibly due to the cumbersome process of isolation and regeneration fromprotoplasts. Also, regeneration from protoplasts can sometimes lead to somaclonal variation with effects on plantdevelopment, which needs to be taken into consideration during both research and breeding projects. A benefitof using genome editing compared with more traditional gene inhibition technologies is that full knockout of anenzyme can be ensured. However, complete loss of an enzymatic activity can occasionally yield unwanted second-ary effects. Hence, partial enzymatic inhibition or a translated inactive enzyme might be beneficial for some traitdevelopments. This can be obtained by combining designed sgRNA targeting with the selection of desiredmutational allelic combinations after analysis (i.e., selecting events with only one to three mutated alleles of atetraploid potato) or by selecting in-frame or tailored mutations with specific amino acid deletions or changes.DSB- and NHEJ-based mutations have limitations since they are randomly composed, often small inserts ordeletions (indels). Furthermore, exact design of the outcome of a mutation would further improve the specificityand enhance the use of the methods to tailor regulating elements or target encoding active sites, or to add a stopcodon to secure loss of enzymatic function. The development of CRISPR-based methods that result in tailoredpoint mutations is ongoing for potato, but they currently have a lower frequency of induced mutations. In thiscontext, DSBs in combination with HR [3], using a template for designed repair, as well as BE [6,13] and primeediting (PE) [14] have been published. Further development of the methods will be important tasks to broadenthe use of genome editing in potato from mainly providing knockouts to also being used for designed knock-insand base substitutions.Trends in GeneticsOPEN ACCESSscreened for. In one of the primary studies,point mutations were induced in ALSby homologous recombination (HR),where the TALEN- and CRISPR-Cas9components and a template for repairwere expressed using a geminivirus repli-con system and Agrobacterium-mediatedtransformation [3]. Increased tolerance tothe herbicide was found in both TALENand CRISPR lines treated with imazamox,which was calculated based on tissueweight compared with wild-type plants, 4weeks post-spraying.Potato with improved starch qualityNative starch comprises two glucose poly-mers, an essentially long-chain linear amy-lose and a highly branched amylopectin.For potato, no genetic variation throughspontaneous mutations has been foundthat would affect the ratio of the two mole-cules of approximately 1:4. CRISPR-Cas9based mutations have been induced instarch biosynthetic genes, altering the ratioof the molecules and the quality of thestarch. A pure amylopectin starch has ad-vantages over native starch as it facilitatesmore sustainable downstream processingwith decreased use of chemicals andenergy. For the development of this trait, aGRANULE BOUND STARCH SYNTHASE(GBSS) gene was mutated using atransgene-free DNA method on potatoprotoplast [4]. During the study, it wasfound that a fraction of the events containedundesired inserts from theDNA vector used.The introduction of the same trait was soonrepeated with CRISPR-Cas9 componentsdelivered as ribonucleoprotein (RNP)complexes, which eliminated the insertionof unintended DNA when synthetically pro-duced RNA was used [5]. Another strategyto develop an amylopectin potato wasdescribed soon afterwards, where the useof base editing (BE) to alter the catalyticregion of the GBSS enzyme was foundsuccessful [6]. Amylopectin starch is mostlikely the most progressed trait developedwith genome editing in potato, and non-transgenic amylopectin lines are currentlygrown in the field for selection and seedmultiplication.A starch with long glucose chains and alow degree of branching, such as amy-lose, can be classified as resistant starchand can yield a low glycemic index infood products, as well as having improvedfilm-forming properties compared withnative starch. The degree of branchingand chain-length distribution of starchwas altered using CRISPR-Cas9-inducedmutations in one or both of two STARCHBRANCHING ENZYME genes (SBE1 andSBE2). In a first study, both stable trans-formation and PEG-mediated protoplasttransfection were used [7]. Mutations inthe SBE2 gene alone gave a 15% de-crease in the degree of branching, whiletetra-allelic mutations in both SBE1 andSBE2 resulted in a more significant de-crease, down to half that of wild-typestarch. In a subsequent study, RNP trans-fection of protoplasts was used, and re-generated potato genotypes containingstarch without any detectable branchingand essentially free of amylopectin whenall eight alleles had indels introduced[8]. In both studies, at least one allele inone or both of the genes was an in-framemutation, and complete loss of bothenzymes was speculated to detrimentallyreduce vitality.Potato with improved tuber qualityPotatoes are most often stored cold forincreased shelf life, which induces starchdegradation and sweetening. When tu-bers are processed at high temperatures,reducing sugars reacts with free aminoacids causing browning and form thecarcinogenic compound acrylamide. Usingtransient expression of TALEN in isolatedprotoplasts, mutations were targeted to avacuolar invertase gene (VInv) that catalyzesthe splitting of sucrose into fructose andglucose. In chips produced from potatoesharvested from a tetra-allelic mutated line,the acrylamide levels were found to be73% reduced compared with wild type [9].Enzymatic browning is a problem forpotato incurred during harvest and post-harvest handling, resulting in the lossof nutritional quality, taste, and texture.The dark-colored pigments are productsof an oxidation reaction catalyzed byPOLYPHENOL OXIDASE (PPO) enzymes.Trends in Genetics, March 2022, Vol. 38, No. 3 219Box 2. Breeding strategies to increase late blight resistanceLate blight, caused by the oomycete Phytophthora infestans, is a devastating disease and requires the most extensive and costly pest management program duringpotato cultivation. Introducing resistance to P. infestans is therefore often high on the wish list in potato breeding. In Figure I, examples of different breeding strategiesand potential future strategies to increase resistance, like traditional crossbreeding, traditional genetic modification, and the evolving genome editing methods andstrategies are shown.Resistance genes (R-genes) Suscepbility genes (S-genes)Crossings with variees or wild relaves or containing R-genesTransfer of R-genes through transformaonR-gene upgrade by rewring exisng genes using genome edingS-gene Knockout through genome edingRandom mutagenesis through chemical or radiaon-treatmentsGene inhibion of S-genes through transformaonTransgenic Cisgenic Non-transgenicTailored mutagenesis of S-genes through genome edingnoitcnufeneGygo lod ohteMem oct uO0102030Traitintrogression(years)Trends in GeneticsFigure I. Examples of breeding strategies to increase late blight resistance in potato. Two main target strategies are displayed – adding or modulatingresistance genes (R-genes) and knocking out, inhibiting, or modulating susceptibility genes (S-genes) – that might be used both individually and in combination tomake the plants more resistant to the disease. Based on the breeding method used, the times for trait introgression differ substantially and the outcome will be atransgenic, non-transgenic, or cisgenic plant that might be differently regulated based on the technique and where in the world it will be cultivated.Trends in GeneticsOPEN ACCESSThe PPO-encoding genes belong to a familythat is differently expressed in various tis-sues. Currently, browning can be managedusing chemical or physical post-harvesttreatments. To address this problem,CRISPR-Cas9 through RNP and proto-plast transfection was used to inducemutations in a PPO2 gene, encoding theprotein accountable for the largest portionof PPO activity in tubers [10]. By inducingtetra-allelic mutations, enzymatic browningwas decreased to approximately 25% ofthe wild type.Sterol glycoalkaloids (SGAs) are toxic com-pounds synthesized in the entire potatoplant, and for safety reasons an upperlimit of 200 mg/kg potato tubers (freshweight) has been set in many countries.The basic level of SGAs varies among vari-eties and can increase through biotic stressor harvest and post-harvest conditions,220 Trends in Genetics, March 2022, Vol. 38, No. 3such as wounding and light exposure of tu-bers. By inducing mutations in a STEROLSIDE CHAIN REDUCTASE 2 (SSR2) gene,using Agrobacterium-mediated transforma-tion of a TALEN [11] and later a CRISPR-Cas9 expression cassette [12], SGA levelswere found to be reduced. In the TALENstudy, a transformant with tetra-allelic muta-tions had reduced SGA levels to 10% of thewild type in leaves. In the CRISPR study, theeffects differed between tissues and eventsanalyzed but were most evident in leaves,where a decrease of up to 56% was ob-served. The majority of the events also hadlower levels in the potato tuber peel of upto 46%.Concluding remarksUsing genome editing methods such asTALEN and CRISPR, genetic variation cantoday be efficiently introduced in potatowhere natural variation is insufficient. Thishas now enabled the cumbersome potatobreeding process to be accelerated in im-provements of specific traits in potato inan already good genetic background. Theexamples given here are traits with healthbenefits, such as lowering toxic com-pounds and improving potato, and starchquality, or traits that tackle environmentalconcerns, like food loss, use of pesticides,and the use of chemicals in downstreamprocesses. Although the traits describedhere are numerous and broad in applica-tion, they represent only a first few exam-ples of what these techniques can do tocontribute to future safe and sustainablefood production.Declaration of interestsNo interests are declared.1Department of Plant Breeding, Swedish University ofAgricultural Sciences, SLU Alnarp, Box 190, 234 22 Lomma,SwedenTrends in GeneticsOPEN ACCESS2Department of Plant Protection Biology, Swedish University ofAgricultural Sciences, SLU Alnarp, Box 190, 234 22 Lomma,Sweden*Correspondence:mariette.andersson@slu.se (M. Andersson).https://doi.org/10.1016/j.tig.2021.10.004© 2021 The Author(s). Published by Elsevier Ltd. This is an openaccess article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).References1. Kieu, N.P. et al. (2021) Mutations introduced in susceptibilitygenes through CRISPR/Cas9 genome editing confer in-creased late blight resistance in potatoes. Sci. Rep. 11, 44872. Hegde, N. et al. (2021) The caffeoyl-CoA O-methyltrans-ferase gene SNP replacement in Russet Burbank potatovariety enhances late blight resistance through cell wall re-inforcement. Plant Cell Rep. 40, 237–2543. Butler, N.M. et al. (2016) Geminivirus-mediated ge-nome editing in potato (Solanum tuberosum L.) usingsequence-specific nucleases. Front. Plant Sci. 7,10454. Andersson, M. et al. (2017) Efficient targeted multiallelicmutagenesis in tetraploid potato (Solanum tuberosum)by transient CRISPR-Cas9 expression in protoplasts.Plant Cell Rep. 36, 117–1285. Andersson, M. et al. (2018) Genome editing in potato viaCRISPR-Cas9 ribonucleoprotein delivery. Physiol. Plant.164, 378–3846. Veillet, F. et al. (2019) The Solanum tuberosum GBSSIgene: a target for assessing gene and base editing in tet-raploid potato. Plant Cell Rep. 38, 1065–10807. Tuncel, A. et al. (2019) Cas9-mediated mutagenesis ofpotato starch-branching enzymes generates a range oftuber starch phenotypes. Plant Biotechnol. J. 17,2259–22718. Zhao, X. et al. (2021) Amylose starch with no detectablebranching developed through DNA-free CRISPR-Cas9mediated mutagenesis of two starch branching enzymesin potato. Sci. Rep. 11, 43119. Clasen, B.M. et al. (2016) Improving cold storage and pro-cessing traits in potato through targeted gene knockout.Plant Biotechnol. J. 14, 169–17610. González, M.N. et al. (2020) Reduced enzymatic browningin potato tubers by specific editing of a polyphenol oxidasegene via ribonucleoprotein complexes delivery of theCRISPR/Cas9 system. Front. Plant Sci. 10, 164911. Sawai, S. et al. (2014) Sterol side chain reductase 2 is akey enzyme in the biosynthesis of cholesterol, the com-mon precursor of toxic steroidal glycoalkaloids in potato.Plant Cell 26, 3763–377412. Zheng, Z. et al. (2021) Editing sterol side chain reductase 2gene (StSSR2) via CRISPR/Cas9 reduces the total steroi-dal glycoalkaloids in potato. All Life 14, 401–41313. Zong, Y. et al. (2018) Efficient C-to-T base editing in plantsusing a fusion of nCas9 and human APOBEC3A. Nat.Biotechnol. 36, 950–95314. Veillet, F. et al. (2020) Prime editing is achievable inthe tetraploid potato, but needs improvement. bioRxivPublished online June 18, 2020. https://doi.org/10.1101/2020.06.18.159111Trends in Genetics, March 2022, Vol. 38, No. 3 221

Potato trait development going fast-forward with genome editing

https://pub.epsilon.slu.se/27336/1/hofvander_p_et_al_220318.pdf

Potato trait development going fast-forward with genome editing

Abstract

Similar works

Full text

Available Versions

Epsilon Open Archive