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Genome Editing in Potato Using CRISPR/Cas Technology: Applications and Challenges

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Deepa Beniwal, Shivani Chauhan and Harnoor Kaur Dhillon

Submitted: 21 November 2023 Reviewed: 22 November 2023 Published: 27 August 2024

DOI: 10.5772/intechopen.1003940

Genetically Modified Organisms IntechOpen
Genetically Modified Organisms Edited by Huseyin Tombuloglu

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Genetically Modified Organisms [Working Title]

Huseyin Tombuloglu and Guzin Tombuloglu

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Abstract

After rice and wheat, potato is the third most important food crop for human consumption. In Europe and several parts of America, potato is consumed predominantly. Like other vegetable crops, potato is prone to several biotic and abiotic stresses and due to climate change, such stresses are getting worse and affecting the yield and quality of harvested product. Both conventional breeding and transgenic approaches are being utilized to enhance the crop production by protecting the crop for different biotic and abiotic stresses. Genome editing technologies such as ZFNs and TALENs were earlier utilized for crop improvement. But recently, RNA-guided nuclease called CRISPR technology is in use for crop improvement. In potato, CRISPR/Cas is utilized for phenotyping, tuber quality, late blight resistance, potato virus Y resistance, herbicide tolerance, starch quality and biosynthesis, enzymatic browning, phosphate transport to roots and several other desirable traits. In this chapter, we summarize the information about major genome editing approaches and use of CRISPR/Cas in potato genome editing.

Keywords

  • conventional breeding
  • transgenics
  • ZFN
  • TALEN
  • CRISPR/Cas
  • biotic and abiotic stress resistance
  • tuber quality and browning
  • starch content

1. Introduction

Potato (Solanum tuberosum), a member of the Solanaceae family, is at fourth position among the most important staple food crops in the globe succeeded by rice and wheat in terms of human consumption [1]. By 2050, the estimated world population will be 9.7 billion and potato will have a vital role in the future in securing food resources for the human beings [2]. From breeding point of view, potato has both morphological and genetical advantages and disadvantages. As potato is asexually propagated, it is exempted from the need to be bred from true seeds to produce homogenous plants. Being tetraploid makes it extremely difficult for desired features to be passed on to offspring in the future [3]. Moreover, potato research is complicated and time-taking process as it is highly heterozygous and polyploid crop, which cause significant hinderance while utilizing standard breeding approaches, ultimately making use of genome editing indispensable [4, 5]. At present, research is being conducted for improvement of specific desirable traits in several crops through genome editing (GE) technologies. Different genome editing approaches are considered as revolutionary for crop improvement as gene knockout and insertion/deletion mutagenesis are possible through genome editing technologies [6]. Crop improvement using genome editing technologies come up with multiple possibilities, such as:

Alteration in only one or two nucleotides among billions obtained from genome of living cells

  • Alteration in whole alleles

  • Insertion of a non-existing gene in the targeted region of the genome.

  • Due to high accuracy of genome editing tools, these are preferred over traditional plant breeding approaches and standard genetic engineering approaches. Along with enhancing the nutritional profile of crops, gene editing tools can effectively be used for incorporating biotic and abiotic stress resistance/tolerance and can develop crop varieties that survive well in adverse climatic conditions such as arid climate [7]. Therefore, gene editing tools can be effectively used for securing the world’s food supply.

Genome editing tools permits double-stranded breaks (DSBs) at particular genomic locations and recovers them using inherent DNA repair mechanisms, such as non-homologous end joining (NHEJ) or homologous recombination (HR). Earlier, this process was assisted by using protein-guided nucleases including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). But lately, the RNA-guided nuclease known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated—has captured the researchers’ attention (Cas) [8]. Both ZFNs and TALENs requires more time and technical expertise than CRISPR/Cas. Numerous significantly important CRISPR/Cas studies have been conducted in potato, here we are featuring some selected achievements of CRISPR-based editing in potato (Figure 1 and Table 1). This chapter provides the current status of CRISPR/Cas, future perspectives, and challenges in potato.

Figure 1.

Advances in potato improvement using clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing.

Target geneDelivery methodEditing agentPhenotypeReference
Starch quality
GBSSIProtoplastCas 9Decrease in amylose content[9]
GBSSIA. tumefaciensCas 9, PmCDA1-CBEN/A[10]
GBSSIA. tumefaciensCas 9Decrease in amylose content[11]
SBE1, SBE2ProtoplastCas 9Decrease in amylose content[12]
Browning
PPO2ProtoplastCas 9Reduced browning[13]
PPO2Protoplast,
A. tumefaciens		Cas 9Reduced browning[13]
Steroidal glyalkaloid (SGA)
SSR2A. tumefaciensCas 966% of WT tuber[14]
Biotic stress tolerance
eIF4E1ProtoplastCas 9Partial resistance to PVY[15]
RNase IIIA. tumefaciensCas 13Improved resistance to sweet potato virus disease[16]
Abiotic stress tolerance
MYB44A. tumefaciensCas 9N/A[17]
Herbicide tolerance
ALS1A. tumefaciensPrime Editor 2Improved herbicide resistance[18]

Table 1.

CRISPR/Cas applications in potato genome editing.

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2. Key genome editing approaches

2.1 ZFNs (Zinc-finger nucleases)

ZFNs were first created by Kim and colleagues [19]. ZFNs develop as a result of the interaction between DNA-binding and DNA-cleaving domains [20]. These synthetic proteins were created based on the fact that the natural restriction enzyme FokI exhibits unique binding and cleaving capabilities [21]. The binding domain consists of eukaryotic transcription factors and 3–6 zinc finger repeats which recognize between 9 and 18 base pairs [22], whereas the DNA-cleavage domain has a FokI restriction enzyme DNA cleavage. To cleave the DNA, the zinc finger domain fused at its C-termini via a peptide linker to the FokI of the cleavage domain. This cleavage domain must dimerize in order to cut DNA. For two cleavage domains to dimerize, the two different ZFNs with their C-termini must bind to opposing DNA strands. If zinc finger, domains are unable to target their specific site within the genome, then off-target cleavage occurs. This off-target cleavage may result in significant double-strand breaks that stop the repair process, which results in cell death or chromosome rearrangements. Further, this leads to the random integration of donor DNA [23]. These domains can be customized to target desired DNA sequences within the complex genomes. By utilizing endogenous DNA repair mechanisms, ZFNs can be used for the precise genome alteration of higher organisms. The efficiency of a given ZFN pair depends on its binding affinity and sequence specificity, both of which impact long-term stability and on-target modification [24]. It is a time taking process to design successful ZFNs. Moreover, ZFNs have poor targeting density which leads to off-targets.

2.2 TALENs

Transcription activator-like effector nucleases (TALENs) are synthetic restriction enzymes that merge the FokI nucleases with the DNA-binding domain of TALEs. TALEs are the natural lethal proteins derived from the Xanthomonas bacteria that bind with specifically targeted DNA sites via a DNA-binding domain in the centre to activate host gene expression. This central domain uses a unique DNA-binding mechanism that involves one-to-one correspondence between an individual repeat and a single nucleotide. Each TALE array has fifteen to nineteen individual tandem repeats and is highly conserved that varies only at amino acid positions twelve and thirteen, known as repeat variable di-residue (RVD) [25]. The RVDs determine the binding specificity of DNA with the TALE array base on one-to-one correspondence along with the base as specified RVD. There are twenty-five types of natural RVDs with good binding specificity, among which NI (Asn-IIe), HD (His-Asp), NH (Asn-Gly), specific for identifying adenine (A), cytosine (C), guanine (G) and thymine (T) [25, 26]. The binding specificity of TALENs is affected by cell type, target sites, duration of effect and delivery system used. The coding for the recognition of the TALENs sequence is comparatively easy, which is an advantage with respect to its targeting density when compared with ZFNs [27]. It allows genome editing with more specificity and low cytotoxicity.

2.3 CRISPR/Cas

CRISPR/Cas is a potent tool for genetic manipulation that precisely mutates specific DNA sequences [28]. There are different CRISPR/Cas systems that differentiate on the basis of the nuclease effector used. This diversity divided the CRISPR/Cas systems into two classes (single/multi-subunit) based on the structure and into six types and 27 subtypes on the basis of the functions of each subunit [29, 30]. It has two components: the Cas9 nuclease and sgRNA. The Cas9 nuclease has two lobes viz., REC for recognition and NUC with nuclease activity. The non-coding sgRNA is complementary to the target sequence (protospacer) of 20 base pairs. This protospacer has a three-base sequence at its 3′ end called PAM (Protospacer Adjacent Motif), recognized by the NUC lobe to produce double-strand breaks (DSB). The DSBs are created when sgRNA/Cas complex binds with the target DNA sequence. Further, these DSBs are repaired by NHEJ (Non-homologous end-joining) and HDR (Homologous directed repair) mechanisms [31].

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3. Genome editing in potato

Introduction of genes related to economically important traits from wild species is very difficult and time intensive assignment in potato (Solanum tuberosum) is as it is a heterozygous polyploid crop. Due to this, traditional breeding approaches cease to work properly when numerous traits and/or new traits which are not available in gene pool need to be introgressed for crop advancement. As genome sequencing data of potato is accessible from public database and standard genetic modification and regeneration protocols are also obtainable, which made potato a prime candidate for gene editing. Thus, gene editing approaches can be used for potato improvement by improving the production and quality characteristics of the crop without affecting maximum allele combination in commercial cultivars [32, 33, 34, 35, 36]. In tetraploid potato, the earliest successful utilization of TALENs were reported for knocking out alleles related to sterol side chain reductase-2 (StSSR2) [37], which are responsible for production of anti-nutritional sterol glycoalkaloid (SGA) [38, 39]. In 2015, it was reported that genome editing approaches such as TALENs [40] and CRISPR/Cas [41] can be utilized to precisely alter the potato genome. CRISPR/Cas9 mediated targeted mutation of StIAA2 gene, encodes for Aux/IAA protein, resulted in homozygous mono and biallelic mutations in the first generation of modified plants [41].

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4. Trait improvement in potato using CRISPR/Cas technology

4.1 Disease resistance

Different biotic stresses are the root cause behind major obstruction in potato production, which ultimately results in enormous loss to farmers. Even now, several scientists are trying to make a key advancement in development of biotic stress resistant potatoes with the help of genome editing approaches. CRISPR/Cas technology of genome editing is considered as an effective substitute for advancing potato breeding programmes.

Phytophthora infestans cause late blight disease of potato, which is the key hinderance in enhancing potato production [42]. Therefore, many researchers are emphasizing on the development of late blight resistant potato cultivars, which can be achieved through knocking out or deleting susceptible genes (S-genes) of late blight [43]. At present, fungicidal sprays and breeding methods are used for the management of late blight in potato. At a recent time, late blight resistance in potato was commenced through knocking out of susceptible genes StDMR6-1 and StCHL1 [44] and Caffeoyl-CoA O-methyltransferase (StCCoAOMT) [45]. Extracellular receptor protein ELR (elicitin response) is found in Solanum microdontum, a wild potato species. Du and co-workers [46] documented utilization of ELR protein for identification of an elicitin which is considered as an extremely conserved in Phytophthora species contributing a wide range durable resistance against late blight pathogen.

For production of potato virus Y (PVY) resistant potatoes, four viral genes, P3, CI, Nib, and CP were targeted through CRISPR/Cas approach. Cas13a protein was utilized to confer resistant against three strains of RNA virus, PVY [47]. Makhotenko and co-workers [48] demonstrated efficient use of host genes (eukaryotic translation initiation factor eIF4E and coilin) for development of PVY resistant potatoes.

4.2 Resistance/tolerance to abiotic stresses

Abiotic stresses include high and low temperatures, drought and salinity cause major constraints in potato production; still limited research work is documented until now. In 2017, Zhou et al. [17] modified MYB transcription factor gene (StMYB44), responsible for restraining StPHO1 gene expression which negatively control phosphate transport activity in potato, with a proficiency rate of 85%. In 2020, Tiwari et al. [49] documented the utilization of gene editing approach CRISPR/Cas for modification of nitrogen metabolism genes to enhance the nitrogen use efficiency of potato plants.

4.3 Herbicide resistance

Butler and co-workers [50] developed a ss gemini virus-based DNA replicon (GVR) as a vector for transfer of TALEN genes and a mutated fragment of ALS1 gene. TALEN genes were transferred to target Acetolactate synthase1 (ALS1) gene in potato while the mutated ALS1gene fragment verified tolerance for numerous categories of ALS-inhibiting herbicides.

4.4 Post harvest

Potato is harvested annually which makes it imperative for producers and sellers to store these tubers in cold stores. Cold storage reduces sprouting and extend shelf life. But during storage in cold storage houses the sucrose in tubers is reduced to sugars which react with amino acids on heating and cause the processed potato products to turn brown and bitter. Moreover, the acrylamide level also goes up. This deterioration in organoleptic properties and enhancement in acrylamide levels negatively affects potato tuber quality thereby leading to a reduced public demand. Accretion of reducing sugars through cold induced sweetening is swayed by several metabolic processes viz starch synthesis, degradation, glycolysis, hexogenesis and mitochondrial respiration. A family of ubiquitous enzymes termed invertases degrades starch into sucrose and fructose [51]. These invertases have been sub-localized to cell wall, vacuole and cytoplasm. Vacuole localized invertases (VInv) play a significant role in cold induced sugar production. Therefore, these have been targeted most to prevent cold induced sweetening. Silencing VInv gene has been accomplished mostly by RNAi [52], TALEN [23] and CRISPR/Cas technologies [53]. Significant reduction in sugar levels were observed in the transgenics developed via these technologies.

4.5 Starch

Potato is a valuable source of starch. Potato starch is used for both culinary and industrial purposes. It is often made up of amylose (20–30%) and amylopectin (70–80%) [54]. The amylose/amylopectin ratio influences the characteristics of both dietary and industrial starch [55]. Amylose-free starch has better freeze-thaw stability and is thus a key component in the manufacturing of frozen foods. Amylopectin has good binding properties and is therefore employed in the paper and glue industries [56]. Therefore, to increase amylopectin content; earlier the genetic engineering efforts primarily aim to produce amylose-free (waxy) potatoes. This might be accomplished by targeting specific genes involved in starch production [57]. Potatoes that produce amylopectin starch may be created by knocking out or silencing the GBSS gene. Various methods have been used to accomplish development of amylopectin rich cultivars viz., radiation-induced mutagenesis, antisense technologies like RNAi, TALENs, and, more recently, CRISPR/Cas [58]. Amylose-free or amylose-reduced lines of sweet potato cultivars have also been developed utilizing CRISPR/CAS genome editing system [59].

Nowadays main focus of researchers has been on altering starch properties to reduce its harmful effects on humans prone to obesity and diabetes. Potatoes with less digestible starch i.e., resistant starch has been engineered by RNAi mediated silencing of genes encoding for starch binding enzymes (SBE). Essentially identical results have been achieved by different means of reducing expression of both SBE isoforms (SBE1 and SBE2): antisense RNA [60], expression of single-domain SBE-specific camelid antibodies [61] and CRISPR/Cas [12, 62]. Though genome editing technologies proved useful in lowering or boosting starch content in potatoes but transient application of vectors harboring inserted DNA fragments lead to the unwanted insertions of vector genome into targeted regions [63].

4.6 Steroidal glyalkaloid (SGA)

Solanaceous plants produce secondary metabolites called glycoalkaloids. More than 80 steroidal glyalkaloids have been identified in different potato species; the two most common ones in cultivated potatoes are α-solanine and α-chaconine [64]. Higher levels of steroidal glyalkaloids have neurotoxic and anti-nutritional effects on human [65]. Therefore, the amount of steroidal glyalkaloids in tubers meant for eating should not exceed the threshold value of 200 mg/kg fresh weight. Nonetheless, significant efforts have been made to reduce SGA content. RNAi suppression of the SGA biosynthetic genes sterol side chain reductase 2 (StSSR2) [26] and GLYCOALKALOID METABOLISM 1 (GAME1) [66] resulted in decreased SGA content. One study on the CRISPR/Cas system’s use for controlling SGA levels in potatoes looked at knocking down the gene encoding steroid 16-hydroxylase (St16DOX), which appears in the genome in a single copy [67]. Two mutants showed positive results for no detectable levels of solanine or chaconine but the plants as a whole were not regenerated so success for CRISPR/Cas-mediated deletion of St16DOX was partial and not all attempts at SGA reduction in potato were equally successful [67]. CRISPR/Cas9-editing of StSSR2 was used to manipulate the SGA level in the cv. Atlantic with significant success [14]. But on the other hand, SGAs are the plant’s first line of defense against viruses and herbivores and participate in ecological interactions with microorganisms therefore eliminating SGAs would be damaging to the plant.

4.7 Enzymatic browning

High tuber quality is a desirable characteristic in the potato processing business and among consumers. Enzymatic browning (EB) causes nutritional quality loss and alters the flavor and texture of tubers. Mechanical injury caused when tubers are cut, sliced or peeled disrupts subcellular compartmentation, resulting in the release of amyloplast-localized PPOs and vacuole-localized phenolic compounds [68]. PPOs catalyze the oxidation of monophenols and/or o-diphenols to o-quinones in the presence of oxygen, which then polymerize and form complexes with proteins, resulting in brown pigment build-up [69]. This enzyme is coded by a multi-gene family, and five PPO genes in potato have been recognized thus far, with numerous allele variants for each gene [68]. Though enzymatic browning is a quantitative characteristic, PPOs are frequently used as target genes to minimize browning in tubers because the PPO loci have a strong correlation with the QTL associated with browning of tubers [70]. Because PPOs are involved in numerous physiological functions as well as defense against diseases and pests, knocking out all PPO genes would almost certainly be fatal to plants. As a result, specific gene targeting or allele silencing is necessary [68]. Substantial progress has been made in this direction. StPPO2 gene was effectively modified in the cv. Desiree via CRISPR/Cas9 [13]. Two different types of edited lines were created by the four-allele StPPO2 edition: those with 111-bp non-frameshift alterations and those with frameshift mutations in the coding region that may have affected the activity of the enzyme after translation. No matter the mutation type, all lines had lower PPO expression levels and were less susceptible to EB than the wild type [13]. GM potato varieties (Innate® potato) with decreased enzymatic browning (EB) and minimal acrylamide generation have been successfully developed and are under cultivation [71].

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5. Challenges in potato gene editing

Potato is an asexually propagated, heterozygous and polyploid crop, which causes multiple obstacles in utilization of genome editing approaches. Major obstacles are: complications in target designing for gene editing and acquiring homozygous mutants with mutation of all targeted gene(s), which make it essential to screen a vast number of transformed plants to identify and propagate mutated lines carrying multiple alleles. Additionally, not each and every potato variety is responsive to transformation. Protoplast mediated transformation and regeneration of plants from leaf as an explant can also cause somaclonal variation, ultimately negatively affecting plant development [8].

To understand complicated economically important traits, breeders are making efforts for development of diploid lines of potato. Self-incompatibility (SI) causes the significant obstruction in development of potato inbred lines, as SI impede in fixing of gene edits and selection of progeny by segregating out the inserted gene. Ye and co-workers [72] used CRISPR/Cas9 approach for production of diploid self-compatible lines of potato by knocking out the Stylar ribonuclease gene (S-RNase), responsible for self-incompatibility in potato. Yet a number of diploid, SI potato lines shows recalcitrant response to transformation through traditional Agrobacterium tumefaciens [73]. To avoid this, Butler and co-workers [73] used A. rhizogenes for speedy production of stable mutants within hairy root clones in potato genotypes which were already showing recalcitrant nature to A. tumefaciens. But in this method, analysis of hairy root clones was the major drawback. Genome editing approach CRISPR/Cas9 was implemented for targeting phytoene desaturase (StPDS) gene in potato. This gene is found in hairy root clones of potato. This method resulted in 64–98% mutation in transformed hairy root clones.

Production of off-target mutants in non-target gene(s) in potato while genome editing is an additional area to be studied. Off-target mutants cause unacceptable alterations in plants and also it complicates the procedure of mutant analysis. Synthetic proofreading Cas9 variants [74], test of sgRNA activity and developing good design [75] are the approaches used to decrease or even remove these off-target mutants.

Also, it is important to produce transgene or foreign gene free potato. To be adopted by consumers and accepted by policy makers, gene edited (GE) crops should not contain any traces of foreign DNA [76].

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6. Conclusion

Genome editing can perform a key role in potato improvement by enhancing the tuber starch content, developing resistance/tolerance against biotic/abiotic stresses and reducing antinutritional factors and toxic compounds. As genome editing approaches are highly effective and accurate, this escalates the possibilities of improvement of other economically valuable plant characteristics also. In case of potato, it requires much efforts and skills for advancement of plant characters governed by multiple genes as compared to those governed by single gene due to heterozygous polyploid nature and asexual reproduction of potato plants. In spite of all these bottlenecks, significant success has been obtained in potato for few plant characteristics and many of them using gene editing approaches (gene knockout or addition/deletion of gene). Researchers have reported multiplexing of SpCas9 along with protoplast mediated transformation as an ideal choice for potato improvement. Over and above, it is important to aware consumers about difference between genome edited (GE) crops or crop varieties and genetically modified (GM) crops or crop varieties. Efforts are being made to keep genetically edited and genetically modified crops separate, which is important for advancement of gene editing approaches and their success. Collectively, genome editing through CRISPR/Cas is an efficient and precise next generation approach for rapid potato breeding to obtain sustainable crop yield.

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Written By

Deepa Beniwal, Shivani Chauhan and Harnoor Kaur Dhillon

Submitted: 21 November 2023 Reviewed: 22 November 2023 Published: 27 August 2024

© The Author(s). Licensee IntechOpen. This content is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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