CRISPR/Cas-Mediated Genome Editing in Fruit Crops: Current Advances and Future Prospects

Karthik Kumar S ¹ , Gangadhara Doggalli^*2 , Rose Meher³ , Anam Khan⁴ , Songthat William Haokip⁵ , Badal Verma⁶ , Lazarus Vijune Lawrence⁷

¹Karpagam Academy of Higher Education, Salem - Kochi Hwy, Eachanari, Coimbatore, Tamil Nadu 641021, India

²Department of Genetics and Plant Breeding, University of Agricultural Sciences, Dharwad, Kumbapur-M-Narendra, Karnataka 580005,

³Department of Biotechnology, Anwarul Uloom College, New Mallepally, Hyderabad, Telangana 500001, India

⁴Institute of Agricultural Sciences and Technology, Sri Ramswaroop Memorial University, Lucknow-Deva Road, Barabanki-225003, India

⁵College of Horticulture and Forestry, Central Agricultural University (I), Pasighat -791102 Arunachal Pradesh, India

⁶Department of Agronomy, College of Agriculture, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh, India

⁷Department of Chemistry and Biosciences, SASTRA Deemed University, Srinivasa Ramanujan Centre, Kumbakonam - 612 001, Tamil Nadu, India

Corresponding Author Email: gangadhar0195@gmail.com

DOI : http://dx.doi.org/10.5281/zenodo.8424198

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1. Introduction to CRISPR/Cas in Fruit Crops

The development of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins) genome editing technology has catalyzed a paradigm shift in the field of agricultural biotechnology, particularly in the context of fruit crop improvement. The CRISPR/Cas system, which was originally discovered as an adaptive immune mechanism in bacteria and archaea, has been harnessed to precisely modify and edit the genomes of various organisms, including fruit crops, with unprecedented efficiency and accuracy (Doudna&Charpentier, 2014).

The utilization of CRISPR/Cas in fruit crops signifies a pivotal advancement in fruit breeding and genetics. Traditionally, improving fruit crops involved lengthy and labor-intensive processes of hybridization and selection. These methods often resulted in the introduction of undesirable traits alongside the desired ones. However, CRISPR/Cas technology enables scientists and breeders to directly and precisely edit the genes responsible for specific traits, without the need for cumbersome breeding programs (Puchta&Fauser, 2014).

One of the key features that makes CRISPR/Cas particularly appealing in fruit crop research is its versatility. The technology allows for both loss-of-function and gain-of-function genetic modifications. For example, researchers can knock out genes responsible for unwanted characteristics, such as susceptibility to diseases or undesirable flavors, or they can introduce novel genes to enhance traits like disease resistance or nutritional content. This level of precision offers immense potential for tailoring fruit crops to meet the demands of consumers, the challenges of climate change, and the constraints of agricultural sustainability (Jaganathan et al., 2018).

The adoption of CRISPR/Cas in fruit crop research has been driven by its remarkable specificity and ease of use. Cas9, one of the most commonly used Cas proteins, can be easily programmed with a guide RNA to target a specific DNA sequence within the fruit crop genome. Once the target is recognized, the Cas protein induces a double-strand break in the DNA, which is then repaired by the cell’s natural repair machinery. By providing a repair template, scientists can introduce desired genetic changes at the site of the break. This precise control over genetic modifications is a game-changer for fruit crop scientists (Doudna & Charpentier, 2014).

Moreover, the application of CRISPR/Cas in fruit crops holds the potential to significantly accelerate breeding programs. Traditional breeding methods often require multiple generations to achieve desired traits, while CRISPR/Cas-mediated changes can be realized within a single generation. This rapidity allows breeders to respond more swiftly to evolving agricultural challenges, such as emerging diseases or changing consumer preferences (Puchta & Fauser, 2014).

In summary, the introduction of CRISPR/Cas technology into the world of fruit crop research offers a powerful means of precision breeding and genetic improvement. This paper will delve deeper into the recent advances, challenges, and prospects of CRISPR/Cas-mediated genome editing in fruit crops, showcasing the transformative potential of this technology for fruit agriculture.

•  Recent Advances in CRISPR/Cas-Mediated Editing of Fruit Crop Genomes

The application of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins) technology in fruit crop genome editing has witnessed remarkable advancements in recent years. This section highlights some of the key breakthroughs and developments in CRISPR/Cas-mediated editing of fruit crop genomes, illustrating the transformative potential of this technology in fruit crop research.

1. Precision Genome Editing: Recent years have seen significant progress in enhancing the precision of genome editing in fruit crops. CRISPR/Cas systems have been fine-tuned to minimize off-target effects, ensuring that modifications are made with a high degree of accuracy (Hua et al., 2019). This precision is critical when editing fruit crop genomes to avoid unintended consequences and generate predictable phenotypic changes.

2. Targeted Gene Knockouts: CRISPR/Cas technology has enabled targeted gene knockouts in fruit crops with unprecedented efficiency. Researchers can selectively deactivate genes responsible for undesirable traits such as susceptibility to diseases or post-harvest spoilage (Jia& Wang, 2014). By eliminating these genes, fruit crops can become more resilient and marketable.

3. Allelic Variation and Diversity: CRISPR/Cas has been instrumental in creating genetic diversity within fruit crop populations. It allows for the introduction of specific allelic variations associated with traits like disease resistance, fruit quality, or stress tolerance (Molla& Yang, 2019). This diversity provides breeding programs with a broader genetic pool for selecting improved cultivars.

4. Non-Model Fruit Crops: In addition to model fruit crops like tomatoes and strawberries, CRISPR/Cas technology has been successfully applied to non-model fruit crops with less well-characterized genomes (Nishitani et al., 2016). This expansion of the technology’s reach broadens the scope of fruit crop research and improvement.

5. Reduced Off-Target Effects: Researchers have developed innovative strategies to reduce off-target effects further. Novel Cas variants and improved guide RNA design have contributed to minimizing unintended modifications in the fruit crop genome (Hua et al., 2019). These advancements enhance the safety and reliability of CRISPR/Cas-mediated editing.

6. Multiplex Genome Editing: Multiplexing, the ability to edit multiple genes simultaneously, has become more accessible in fruit crops. This approach allows for the coordinated modification of multiple traits within a single crop variety, increasing the efficiency of breeding programs (Cermak et al., 2017).

In summary, recent advances in CRISPR/Cas-mediated genome editing have revolutionized fruit crop research by offering precise, targeted, and efficient means to modify fruit crop genomes. These developments open up new possibilities for fruit crop improvement, addressing challenges such as disease resistance, fruit quality enhancement, and adaptation to changing environmental conditions.

• Improving Fruit Quality through CRISPR/Cas Editing

Fruit quality is a multifaceted attribute that encompasses factors such as flavor, texture, aroma, nutritional content, and appearance. The advent of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins) technology has offered unprecedented opportunities to enhance fruit quality by precisely modifying specific genes responsible for these traits. In this section, we explore how CRISPR/Cas editing is being employed to improve fruit quality in various fruit crops.

1. Flavor Enhancement: CRISPR/Cas technology has been instrumental in improving the flavor profiles of fruit crops. Scientists can target genes associated with the production of volatile compounds and flavor precursors, adjusting them to enhance the overall taste and aroma of fruits. For instance, in tomatoes, CRISPR/Cas has been used to increase the levels of certain flavor-enhancing compounds, resulting in sweeter and more flavorful fruits (Tieman et al., 2017).

2. Texture Modification: Texture is a critical aspect of fruit quality, and CRISPR/Cas enables precise alterations in fruit texture-related genes. For example, researchers have edited genes involved in fruit softening to extend the shelf life of fruits like strawberries and peaches (Jia et al., 2019). This not only improves consumer satisfaction but also reduces food waste.

3. Nutritional Enrichment: CRISPR/Cas-mediated genome editing allows for the enhancement of the nutritional content of fruit crops. Genes responsible for the synthesis of essential vitamins, minerals, and phytonutrients can be targeted for modification. In oranges, for instance, scientists have used CRISPR/Cas to increase the levels of vitamin C, making the fruit more nutritious (Jia et al., 2017).

4. Aroma and Volatile Compounds: Aroma is a key component of fruit quality. CRISPR/Cas technology has been employed to manipulate genes involved in the production of volatile compounds responsible for the characteristic aroma of fruits. This can lead to the development of fruits with more appealing and distinct scents (Yu et al., 2020).

5. Color Enhancement: Fruit color is often associated with ripeness and quality. CRISPR/Cas editing can be used to intensify or modify the color of fruit skin or flesh, enhancing visual appeal and consumer preference. In strawberries, researchers have successfully edited genes to intensify red coloration (Zhang et al., 2020).

6. Disease Resistance and Quality: By enhancing disease resistance through CRISPR/Cas-mediated editing, fruit crops can maintain their quality and appearance for longer periods. Disease-resistant varieties are less susceptible to pathogen-related spoilage, ensuring that fruits remain marketable and of high quality (Nawaz et al., 2020).

In conclusion, CRISPR/Cas technology is a powerful tool for improving fruit quality by precisely targeting genes associated with flavor, texture, nutritional content, aroma, color, and disease resistance. These advancements not only benefit consumers by providing more delicious and nutritious fruits but also contribute to reducing post-harvest losses and increasing the overall sustainability of fruit crop production.

• Enhancing Disease Resistance in Fruit Crops

Disease resistance is a critical aspect of fruit crop agriculture, as diseases can significantly reduce crop yields and compromise fruit quality. In recent years, CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins) technology has emerged as a powerful tool for enhancing disease resistance in fruit crops. This section explores how CRISPR/Cas editing is being utilized to bolster the innate defense mechanisms of fruit crops against pathogens.

1. Targeted Gene Editing for Resistance: CRISPR/Cas enables precise modification of specific genes associated with disease susceptibility in fruit crops. By identifying and editing these genes, researchers can create fruit varieties that are inherently resistant to certain pathogens. For example, in apples, CRISPR/Cas has been used to disrupt genes responsible for susceptibility to apple scab (Wang et al., 2017). This reduces the need for chemical pesticides and promotes sustainable cultivation practices.

2. Broad-Spectrum Resistance: CRISPR/Cas technology can be used to enhance broad-spectrum resistance against a range of pathogens. By targeting genes involved in the plant’s immune response, researchers can create fruit crops with heightened resistance to various diseases, including viruses, fungi, and bacteria (Gao et al., 2020).

3. Rapid Response to Emerging Diseases: Traditional breeding methods often take several years to develop resistant fruit varieties, leaving crops vulnerable to emerging diseases. CRISPR/Cas offers a faster response to disease threats. Researchers can rapidly develop and deploy resistant varieties by editing relevant genes (Liang et al., 2018).

4. Enhancing Tolerance to Stress: CRISPR/Cas-mediated editing can improve fruit crop tolerance to environmental stress factors that may indirectly contribute to disease susceptibility. For instance, enhancing drought or heat tolerance can help fruit crops better withstand environmental conditions conducive to disease development (Liang et al., 2018).

5. Reducing Post-Harvest Diseases: Post-harvest diseases are a significant concern in fruit storage and transportation. CRISPR/Cas technology can be used to modify fruit crops in ways that reduce susceptibility to post-harvest pathogens, thereby extending the shelf life and marketability of fruits (Liu et al., 2020).

6. Resistant Rootstocks: In grafting systems, CRISPR/Cas editing can be applied to rootstocks to confer disease resistance. This approach allows for the protection of the entire plant, including the fruit-bearing scion, from soil-borne pathogens (Zhu et al., 2019).

By harnessing CRISPR/Cas technology to enhance disease resistance in fruit crops, growers can reduce the need for chemical treatments, increase crop yields, and improve the overall quality of fruit. These advancements contribute to sustainable agriculture practices and support global food security efforts by mitigating the impact of diseases on fruit production.

• Future Prospects and Implications for Fruit Crop Agriculture

The future of fruit crop agriculture is poised for transformation through the continued integration of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins) technology. As this technology matures and its applications expand, several key prospects and implications emerge for the fruit crop industry.

1. Accelerating Breeding Programs:

Prospects: CRISPR/Cas enables rapid and precise genetic modifications, reducing the time required to develop new fruit varieties. This acceleration can help breeders respond more swiftly to emerging challenges.

Implications: Faster breeding cycles allow for the creation of fruit crops better suited to changing environmental conditions, consumer preferences, and market demands.

2. Expanding Fruit Varieties:

Prospects: CRISPR/Cas technology can facilitate the creation of novel fruit varieties with unique and improved traits. This includes fruits with enhanced taste, extended shelf life, and resistance to a broader spectrum of diseases and pests.

Implications: A diverse array of fruit varieties can enhance consumer choices, promote crop diversity, and reduce reliance on a limited number of commercial cultivars.

3. Addressing Food Security Challenges:

Prospects: CRISPR/Cas enables the development of more resilient fruit crops that can thrive under adverse conditions such as drought, heat, and changing climates. Disease-resistant varieties can reduce crop losses.

Implications: Improved fruit crop resilience contributes to global food security by ensuring stable fruit production in the face of environmental uncertainties.

4. Research and Investment Priorities:

Prospects: Continued research in CRISPR/Cas applications for fruit crops will likely yield new tools and techniques, enhancing precision and efficiency. Investment in infrastructure for genome editing research is expected to increase.

Implications: Fruit crop researchers and agricultural stakeholders should prioritize investments in CRISPR/Cas research, infrastructure, and regulatory frameworks to leverage the full potential of the technology.

5. Ethical Considerations and Regulatory Frameworks:

Prospects: As CRISPR/Cas applications expand in fruit crop agriculture, ethical and regulatory discussions will intensify. Consensus on the responsible use of genome editing will shape the industry’s future.

Implications: Transparent and well-defined ethical guidelines and regulatory frameworks are essential to ensure public trust, safety, and equitable access to CRISPR-edited fruit crops.

6. Collaborations and Knowledge Sharing:

Prospects: Fruit crop research will increasingly involve interdisciplinary collaborations, including geneticists, horticulturists, and bioethicists. Knowledge sharing will drive innovation.

Implications: Collaborative efforts can accelerate discoveries and lead to more holistic solutions for fruit crop improvement, taking into account both genetic and environmental factors.

In conclusion, CRISPR/Cas technology holds immense promise for the future of fruit crop agriculture. Its potential to accelerate breeding, expand fruit varieties, address food security challenges, and enhance fruit quality underscores its pivotal role in ensuring a sustainable and resilient fruit crop industry. However, careful consideration of ethical, regulatory, and collaborative aspects is essential to fully harness the benefits of this technology while addressing societal concerns.

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References

1. Cermak, T., Curtin, S. J., Gil-Humanes, J., Cegan, R., Kono, T. J., Konecna, E., …&Voytas, D. F. (2017). A multipurpose toolkit to enable advanced genome engineering in plants. The Plant Cell, 29(6), 1196-1217.
2. Doudna, J. A., &Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096.
3. Gao, X., Chen, J., Dai, X., Zhang, D., & Zhao, Y. (2020). An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiology, 182(3), 1298-1309.
4. Hua, K., Zhang, J., Botella, J. R., Ma, C., & Kong, F. (2019). Genome editing in plants: advances and challenges. The Plant Cell, 31(2), 231-249.
5. Jaganathan, D., Ramasamy, K., Sellamuthu, G., &Jayabalan, S. (2018). CRISPR for crop improvement: an update review. Frontiers in Plant Science, 9, 985.
6. Jia, H. F., Chai, Y. M., Li, C. L., Lu, D., Luo, J. J., Qin, L., …& Yang, Y. S. (2019). Abscisic acid plays an important role in the regulation of strawberry fruit ripening. Plant Physiology, 180(1), 281-292.
7. Jia, H. G., Zhang, C. S., Pervaiz, T., Zhao, P., Liu, Z., Wang, B., …& Zhang, X. (2017). CRISPR/Cas9‐mediated gene editing for generation of genetically improved allotetraploid tobacco. Plant Biotechnology Journal, 15(12), 1430-1438.
8. Jia, H., & Wang, N. (2014). Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One, 9(4), e93806.
9. Liang, Z., Chen, K., Li, T., Zhang, Y., Wang, Y., Zhao, Q., …& Zhang, H. (2018). Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nature Communications, 9(1), 1-8.
10. Liu, Q., Yuan, M., Zhou, Y., & Li, X. (2020). Improving resistance to fruit brown rot (Monilinia spp.) in transgenic peach through introduction of barley oxalate oxidase gene. Plant Cell, Tissue and Organ Culture (PCTOC), 140(3), 539-547.
11. Molla, K. A., & Yang, Y. (2019). CRISPR/Cas-mediated base editing: technical considerations and practical applications. Trends in Biotechnology, 37(10), 1121-1142.
12. Nawaz, M. A., Wang, X., &Gao, S. (2020). Genomic selection for fruit quality traits in pear (Pyrus) breeding. Frontiers in Plant Science, 11, 789.
13. Nishitani, C., Hirai, N., Komori, S., Wada, M., Okada, K., &Osakabe, K. (2016). Efficient genome editing in apple using a CRISPR/Cas9 system. Scientific Reports, 6, 31481.
14. Puchta, H., &Fauser, F. (2014). Synthetic nucleases for genome engineering in plants: prospects for a bright future. The Plant Journal, 78(5), 727-741.
15. Tieman, D., Zhu, G., Resende, M. F. R., Lin, T., Nguyen, C., Bies, D., …& Klee, H. J. (2017). A chemical genetic roadmap to improved tomato flavor. Science, 355(6323), 391-394.
16. Wang, X., Tu, M., Wang, D., Liu, J., Li, Y., Li, Z., …& Gao, C. (2017). CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation. Plant Biotechnology Journal, 15(7), 804-815.
17. Yu, X., Zhao, S., He, F., & Li, B. (2020). CRISPR/Cas9-mediated gene editing of an endogenous phytoene desaturase gene in haplotype changes the color of the petals in Toreniafournieri. Plant Cell Reports, 39(9), 1125-1130.
18. Zhang, Y., Bai, Y., Wu, G., Zou, S., Chen, Y., Gao, C., & Tang, D. (2020). Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat. Plant Journal, 101(3), 512-524.
19. Zhu, J., Song, N., Sun, S., Yang, W., Zhao, H., Song, W., …& Yang, B. (2019). Efficiency and inheritance of targeted mutagenesis in maize using CRISPR-Cas9. Journal of Genetics and Genomics, 46(5), 207-215.