Using CRISPR/Cas for Potato Innovation: A Review of Recent Developments and Challenges
by Jorge Luis Alonso with GPT-4
The review, published in the journal Planta, highlights recent advances in the optimization of CRISPR/Cas-based genome editing in potato, specifically addressing the unique biological challenges of this species. It also explores the potential for the development of transgene-free potato varieties. In addition, the review discusses improvements in stress resistance, nutritional value, starch composition, and storage traits. A summary of the main points is provided in the following text.
Introduction
Potatoes, an important cash crop and rich source of nutrition, are grown in 140 countries and are expected to produce 359 million tons in 2020. Native to South America, there are four cultivated and 107 wild potato species.
Despite being a staple crop, potatoes face the daunting challenges of climate change. These include the need for sustainable yield increases and the development of heat-tolerant varieties. It’s important to note that potato varieties respond differently to abiotic and biotic stresses, with many factors affecting productivity, quality and storage.
During the late 20th century, conventional breeding was aimed at improving potato varieties. However, these efforts faced significant challenges due to limited gene pools and complex genetic structures.
In contrast, genetic engineering offers a faster solution by introducing genes for improved resistance, nutrition, and reduced toxicity. To date, several genetically modified potato varieties have been commercialized with varying degrees of success. Yet, consumer acceptance of these varieties remains low.
Recently, new breeding techniques such as CRISPR/Cas technology have been used to improve potatoes. The goal is to produce transgene-free plants and tubers. This review aims to examine the successful use of CRISPR/Cas technology to improve economically important potato traits. Moreover, it will explore the challenges, opportunities and limitations of its application.
Potato functional genomics before CRISPR/Cas
Before the advent of CRISPR/Cas, techniques for functional plant genomics included primarily RNA silencing and genome mutagenesis.
In particular, RNA silencing technologies such as RNAi have been used in potato research. They have been used to generate potato lines with new functional traits and resistance to biotic stresses.
Genome editing strategies in potato include induced mutagenesis. However, this method is somewhat limited by the tetraploid nature of potato. Instead, engineered endonucleases such as zinc finger (ZNF) nucleases and transcription activator-like (TAL) nucleases have emerged as important tools for functional genomics. Interestingly, TALs are more applicable to plants, including potato, and allow for targeted genome modification. In fact, they provide more effective gene knockout than RNAi.
Standard plant transformation methods can be used to deliver TAL constructs to target plants. Furthermore, many reverse genetics solutions can be applied to improve the efficiency of CRISPR/Cas gene editing in potato.
Overview of CRISPR/Cas technology
CRISPR/Cas technology has revolutionized plant research and crop improvement. It’s more affordable and less labor-intensive than ZNFs or TALENs, but more prone to off-target effects.
In practice, both conventional CRISPR/Cas9 systems and engineered Cas enzymes have been used for gene editing in potatoes. Specifically, the Cas9 endonuclease cuts DNA at specific sites guided by a single guide RNA (sgRNA).
Further modifications of Cas9 have led to the development of more precise genome editing technologies such as base and prime editing. Moreover, Cas12a is another Cas protein used to edit AT-rich genomes, enabling multiplex genome editing. Additionally, Cas13, which cuts single-stranded RNA, has been used for RNA knockdown and to enhance plant immunity to RNA viruses.
Despite the challenges posed by the complex biology of potato, researchers continue to work to optimize CRISPR/Cas technology for this crop.
Potato as a target plant for CRISPR/Cas genome editing technology
Potatoes, an important food source and raw material for the food processing industry, can be improved using biotechnology techniques such as genetic engineering and genome editing, including the use of CRISPR/Cas.
These methods reduce the time required to introduce or modify targeted genes. Nevertheless, complex potato genetics, including tetraploid chromosomes and high heterozygosity, present significant barriers to genome editing.
It should be noted that most agronomic traits in potato are quantitative and controlled by multiple genetic factors, making it difficult to achieve desired traits. Besides, somaclonal variation can occur during plant regeneration after genome editing, potentially leading to phenotypic and genotypic differences.
Despite these challenges, it’s clear that CRISPR/Cas technology has immense potential to effectively modify potatoes, particularly through the use of gene silencing and knockout techniques.
Applications of CRISPR/Cas in potato
The application of CRISPR/Cas technology to genome editing in potato follows two main lines of research:
First, one line of research focuses on technical aspects, testing new ideas and implementing effective innovations from other organisms. These advances can then be applied to other tuber and polyploid crops.
Second, another line of research uses CRISPR/Cas techniques to create or enhance phenotypic traits. This often results in genome-edited lines with significant commercialization potential.
Potato as a model plant
There are several advantages to using potato as a model plant. Potato varieties serve as attractive research models due to their ease of cultivation, optimized protocols, and readily available research tools.
Among the tetraploid varieties, Désirée is used for CRISPR/Cas technology studies. However, it’s important to note that lower ploidy potatoes are more suitable for reverse genetics and CRISPR/Cas technology testing.
Most CRISPR/Cas research in potatoes focuses on optimizing techniques that have already been tested in other species. The solutions developed through this research could potentially benefit other tuber and polyploid crops.
Marker (Reporter) genes for CRISPR/Cas testing in potato
Endogenous genes such as ALS and PDS, which have readily detectable phenotypic effects, are often used to test new genome editing techniques in plants.
For example, ALS is involved in amino acid synthesis and can be strategically targeted for herbicide resistance, while PDS plays a role in carotenoid synthesis, with knockout results leading to plant albinism. GBSS serves as another commonly used endogenous reporter in tetraploid potatoes, affecting starch structure.
These marker genes are proving invaluable for several purposes, including monitoring editing efficacy, detecting mutations, selecting for genome-edited events, and generating non-transgenic genome-edited plant lines.
CRISPR/Cas delivery methods in potato
Methods of delivery of CRISPR/Cas components, such as Agrobacterium-mediated transformation and protoplast transfection, significantly affect the efficiency of gene editing in potato.
Both stable and unstable agrotransformation methods can be used, with stable integration often increasing the chances of full allelic mutations. However, it’s worth noting that stable transformation can sometimes lead to unwanted effects such as transgene silencing or random T-DNA integration.
In contrast, transient transformation methods circumvent these problems and allow transient CRISPR/Cas expression, thereby allowing the generation of non-transgenic plants. Proper selection of bacterial strains, binary vectors, and target tissues is essential to improve the efficiency of genome editing.
Optimization of CRISPR/Cas approaches in potato
Early efforts in CRISPR/Cas editing in potato focused primarily on gene function studies and trait improvement, using native RNA promoters and diploid breeding lines to increase the induction of full allele mutants.
GVR-based vectors have shown promise in increasing the efficiency of gene targeting, but require further optimization for use in potato.
In an effort to induce changes in all alleles of a targeted gene, researchers have explored methods that bypass time-consuming crosses and reduce the effects of inbreeding depression.
Studies have shown success in generating tetra-allelic mutants with CRISPR/Cas constructs that target multiple sites. In parallel, other research efforts are focused on enhancing Cas activity and using multiple sgRNAs to increase the efficiency of CRISPR/Cas9 editing.
Testing the latest CRISPR/Cas technology
Base editing (BE) and prime editing (PE) techniques have been tested in various plants, including potato, for precise genome editing.
For example, the A3A-PBE base editor fusion protein has demonstrated high precision and low indel frequency in potato protoplasts. On the other hand, other studies have shown successful BE with high mutation efficiency but also frequent indels, clearly indicating the need for further optimization.
PE strategies applied in potato have shown relatively low editing rates. However, the effect introduction of nucleotide transversions in targeted genes suggests significant potential for future developments in precise genome editing.
Developing strategies for the generation of non-transgenic genome-edited potato
It’s worth noting that public opposition and complex regulatory approval processes for GM crops have driven research to optimize techniques for creating non-GM crops.
In particular, CRISPR/Cas technology offers new prospects for editing existing potato varieties without the need for stable integration of CRISPR/Cas genes.
Transient activation of CRISPR/Cas components in target cells can effectively generate non-GM potato plants, and effective methods have been described for both Agrobacterium-mediated delivery and protoplast transfection. However, there are certain limitations to consider, such as the risk of inducing extensive somaclonal changes in regenerated lines.
Alternative techniques, such as in planta agroinfiltration and virus-induced genome engineering, may also prove valuable for the delivery of CRISPR/Cas components.
Potato as a food and industrial crop
Improvement of potato cultivars for both food and industrial use focuses on tuber yield and quality, parameters that depend on both genotype and environmental conditions.
The integration of genome editing technologies such as CRISPR/Cas with rich potato genetic resources is highly desirable for future breeding strategies.
Efficient improvements mediated by CRISPR/Cas include a variety of tactics. These range from removing self-incompatibility and enhancing resistance to biotic stresses to increasing tolerance to cold-induced sweetening (CIS) and enzymatic browning (EB).
Furthermore, efforts have been made to reduce the steroidal glycoalkaloid content and modify starch composition, which both have significant implications for the quality of potato production.
Generation of self-compatible diploid potato
A revolutionary approach to potato breeding proposes the reinvention of potato varieties as diploid inbred lines propagated by True Potato Seeds (TPS).
In particular, TPS propagation offers several advantages, including the near absence of pathogens, making it useful for reducing costs in developing countries.
Historically, the use of traditional F1 inbred lines, common in other crops, has been limited in potatoes due to their heterozygous tetraploid nature and self-incompatibility.
However, by ingeniously targeting the S-RNase function using CRISPR/Cas9, researchers have been able to create self-compatible diploid lines. This innovation has enabled the production of inbred lines to significantly improve potato breeding and enrich the potato gene pool.
Enhancing biotic and abiotic stress resistance
Phytophthora infestans, a devastating pathogen of potatoes, is a major concern in potato production. While fungicides are commonly used to control late blight, the disease caused by this pathogen, they pose potential environmental risks. Therefore, a more sustainable solution is to develop resistant varieties.
Innovatively, CRISPR/Cas-based technologies have been used to increase resistance in potatoes without the need to modify the R genes. Two strategies that have been used are knockin of metabolite biosynthetic genes and knockout of susceptibility genes. Impressively, both strategies have shown promising results in enhancing resistance.
Similarly, CRISPR/Cas technology has been used to create resistance to the potyvirus PVY, a destructive viral pathogen that poses a significant threat to potato crops. However, the use of this technology to generate abiotic stress-resistant varieties remains somewhat limited.
Reducing cold-induced sweetening
One of the challenges of long-term storage of potato tubers at low temperatures is the degradation of potato chip quality. This degradation is caused by amylolytic enzymes that break down starch into reducing sugars. Specifically, the invertases responsible for this degradation have been identified as potential targets for reducing sugar content in potato tubers.
In response, CRISPR/Cas technology was used to target VInv expression, which is regulated at both the transcriptional and post-translational levels. In a novel approach, targeted DNA methylation was performed using CRISPR-dCas9-DRM2 technology. This process significantly reduced VInv expression, which in turn led to a reduction in sugar accumulation in tubers. Ultimately, this led to an improvement in chip quality.
Increasing tolerance to enzymatic browning
Tuber quality is of high importance to both the potato processing industry and consumers. One factor that can reduce nutritional quality and alter tuber flavor and texture is enzymatic browning (EB). EB typically occurs when tubers are exposed to processing. This exposure results in the release of polyphenol oxidases (PPOs) and phenolic compounds, ultimately leading to brown pigment accumulation.
While chemical approaches do exist to prevent EB, they aren’t always the best solution. This is because PPOs are involved in many physiological processes, and so a more precise method such as gene targeting or allele silencing is often required.
In an effort to address this, researchers have targeted the StPPO2 gene in potato cv. Désirée using the CRISPR/Cas9 system. This resulted in reduced PPO expression levels and, notably, reduced susceptibility to EB when compared to the wild type.
Decreasing the steroidal alkaloid content
Glycoalkaloids are secondary metabolites found in solanaceous plants. The most abundant of these in cultivated potatoes are α-solanine and α-chaconine. The accumulation of these substances depends on both the genotype and the environment. Because of their neurotoxic properties and bitter taste, it’s important that their levels in tubers do not exceed 200 mg kg-1 fresh weight.
Although their complete elimination would be detrimental to the crop, efforts have been made to reduce steroidal glycoalkaloid (SGA) levels. These attempts have included methods such as crossbreeding and RNAi silencing.
More recently, the CRISPR/Cas system has been used to manipulate SGA levels. There have been some successful attempts to knock out the St16DOX gene, resulting in mutant hairy root lines deficient in α-solanine and α-chaconine. However, it’s important to note that not all trials were equally effective. Results in reducing SGA varied among potato varieties.
Modification of starch composition
Potato starch, which is composed of amylose and amylopectin, plays an essential role for both food and industrial purposes. The ratio of amylose to amylopectin significantly influences the properties of the starch, and as a result, genetic engineering strategies often aim to either create amylose-free (waxy) potatoes or increase the amylose content.
In this context, CRISPR/Cas technology has proven to be effective for both approaches. For instance, amylopectin starch, which is utilized in industries such as frozen food and paper, can be produced by knocking out or silencing the gene that encodes GBSS.
In an effort to produce non-transgenic plants, studies have been exploring various methods. These include the transient expression of the CRISPR/Cas system in protoplasts and the delivery of CRISPR/Cas as preassembled RNPs.
On the other hand, increasing the amylose content results in higher levels of resistant starch, which is beneficial for both health and industrial applications. Potato lines with enhanced amylose content were generated by employing RNAi-mediated knockdown of starch branching enzyme (SBE) genes and using CRISPR/Cas for gene editing.
Currently, the transient expression of sgRNA/Cas9 constructs and the delivery of RNP complexes to protoplasts are viewed as promising tools. These methods can potentially generate transgene-free plants with mutations in SBE genes, paving the way for the development of improved potato varieties.
Future prospects
- In the quest to improve the potato, scientists are focusing on increasing its nutritional value, yield and safety. Indeed, CRISPR/Cas technology, precise mutation methods such as PE and BE, holds great promise for achieving these goals. Given the controversy and lack of public acceptance of GM crops, researchers are prioritizing the development of techniques to create non-GM crop varieties.
- Innovative genome editing techniques offer potential for the production of non-GM crops. For example, a recent study in N. benthamiana used a dual-based vector system to co-express the Cas12a nuclease gene and sgRNA, demonstrating the ability to introduce controlled mutations without producing stably transformed lines. Such advances could streamline the commercialization of varieties with desirable traits.
- In addition, CRISPR/Cas is useful for improving traits that are determined by multiple genes. For example, simultaneous gene knockout can improve tuber quality for the processing industry by increasing tolerance to cold-induced sweetening (CIS). Another focus is the reduction of steroidal glycoalkaloids (SGAs) in potato tubers. Precise selection of candidate genes is essential, and CRISPR/Cas-mediated editing of GAME genes offers potential solutions.
- Improvements in potato genome editing techniques will also benefit molecular plant physiology research. CRISPR/Cas tools allow for more precise changes at the genome level, which can lead to a deeper understanding of potato gene function. Improving the precision and efficiency of CRISPR/Cas methods in potatoes can impact areas such as oil accumulation and recombinant protein production.
- One possibility under consideration is to increase the triacylglycerol (TAG) content in potato tubers to improve their nutritional value and suitability for industrial applications. Furthermore, CRISPR/Cas technology can be used to further manipulate carbon flux, shedding light on the relationship between carbohydrate and lipid metabolism in tubers.
- Interestingly, potatoes have been recognized for their potential as a source of recombinant proteins. Temporary storage of tubers containing active recombinant proteins offers a cost-effective solution for the production of therapeutics or vaccines, especially in developing countries. The use of CRISPR/Cas technology to eliminate the multi-gene patatin family could help overcome barriers to recombinant protein production and purification.
- Finally, humanization of post-translational modifications, especially glycosylation, is a critical step in adapting potato tubers for human protein production. CRISPR/Cas methods can generate glycoengineered tubers, increasing the potential for potatoes to be used in molecular pharming and the production of humanized glycoproteins.
Source: Chincinska, I. A., Miklaszewska, M., & Sołtys-Kalina, D. (2022). Recent advances and challenges in potato improvement using CRISPR/Cas genome editing. Planta, 257(1). https://doi.org/10.1007/s00425-022-04054-3
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