Banana including plantain (Musa spp.) plays a pivotal role in global agriculture and food security, provides a reliable source of affordable and nutritious food. With its year-round availability and adaptability to diverse climates in the tropics and subtropics (Jones, 2000), banana emerge as a steadfast source of essential nutrients, particularly potassium, vitamin C, vitamin B6, and dietary fiber (Kumari et al., 2023). It not only provides a swift and convenient energy boost but also assumes a central role as a staple in the diets of millions of people in regions, especially within tropics.

In addition to its nutritional value, the economic significance of banana cannot be overstated. Serving as a critical export commodity for numerous tropical nations, banana contributes substantially to international agricultural trade, with vast plantations spanning over 140 countries and islands, covering over 12 million hectares globally, and 7.5 million hectares in Africa (FAOSTAT, 2021). The undeniable ubiquity and economic impact of banana underscore their pivotal role in sustaining the wellbeing of populations worldwide.

However, despite its importance, banana production faces a multitude of challenges, including biotic and abiotic stresses, declining soil fertility, limited genetic diversity, and insufficient availability of clean planting material, particularly among smallholder farmers. The prevalence and co-existence of various pathogens and pests further exacerbate these challenges, leading to significant yield gaps and threatening the sustainability of banana cultivation in affected regions (Tripathi et al., 2020). In response to these pressing challenges, the application of cutting-edge technologies such as CRISPR/Cas-based gene editing emerges as a promising avenue for revolutionizing banana production. By harnessing the power of gene editing, researchers are developing improved banana varieties with enhanced resistance or tolerance to biotic and abiotic stresses, thereby bolstering productivity and resilience in the face of environmental adversities. This article provides a comprehensive overview of recent advancements and future prospects in the utilization of gene editing technologies for the development of better banana varieties. This article seeks to elucidate the potential of CRISPR/Cas-based gene editing in driving innovation and sustainability within the global banana industry.

Banana production grapples with an array of biotic and abiotic challenges that pose significant threats to both yield and fruit quality. These challenges are multifaceted and contingent upon factors such as geographical location, climate conditions, and specific agricultural practices. Among the notable biotic constraints, bacterial, fungal, and viral diseases stand out as major threats to banana crops.

Particularly menacing are diseases like Fusarium wilt disease, caused by the Fusarium oxysporum f. sp. cubense (Foc) fungus, and black Sigatoka, attributable to the Mycosphaerella fijiensis fungus. These diseases have the potential to inflict substantial yield losses, with Fusarium wilt, in particular, proving to be one of the deadliest biotic constraints (Ploetz, 2015). Fusarium wilt, also known as Panama disease, damages the plant’s vascular system, causing wilting and, ultimately, death. Compounding the challenge is the soilborne nature of Foc, which can persist in the soil for decades, posing significant challenges for disease management (Hennessy et al., 2005).

Furthermore, the emergence of Foc tropical race 4 (TR4) poses a grave threat to banana production globally. This deadly fungal strain has been identified in various regions worldwide, including key banana-producing countries like Mozambique, Colombia, Israel, Jordan, Turkey, Mayotte, Peru, and Venezuela. The spread of TR4 in Africa is particularly alarming, given the continent’s status as the world’s second-largest banana producer and consumer (Viljoen et al., 2020). With limited control, prevention, and management tools currently available, addressing the spread of TR4 remains a critical challenge for sustaining banana production (Ploetz, 2015). The only viable option is disease-resistant banana varieties that ensures increase productivity with high nutritional value by application of genetic engineering (Dale et al., 2017). These TR4 resistant banana are recently approved for environmental release in Australia (OGTR, 2024).

Second, the most important fungal disease is Black Sigatoka, affecting mainly the lower leaves of banana plants and directly reducing the yield of the crop and poor quality of fruits (Arango Isaza et al., 2016).

In addition to fungal diseases, bacterial infections such as banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pv. musacearum, pose significant threats to banana production in Africa. The impact of BXW disease on banana yield losses is particularly severe in the Democratic Republic of Congo (83%), Uganda (71%), and other East African countries like Burundi, Kenya, Rwanda, and Tanzania, where losses range from 39% to 51% (Ainembabazi et al., 2015). Notably, the lack of disease-resistant banana varieties exacerbates the economic repercussions of BXW, with estimated losses ranging from USD 2 to 8 billion over a decade (Abele and Pillay, 2007; Biruma et al., 2007; Tripathi et al., 2009). No cultivated banana varieties have yet to demonstrate resistance except for the wild-type diploid banana Musa balbisiansa, which is native to Southeast Asia (Nakato et al., 2019).

Several viruses, including banana streak virus (BSV, genus Badnavirus) and banana bunchy top virus (BBTV, genus Babuvirus) also affect banana production worldwide because of their effects on yield, quality, and limitations to the international germplasm exchange due to presence of viruses in planting materials, posing a severe threat to food and nutrition security in banana-growing regions (Kumar et al., 2015).

Banana also face pressure from various pests, including aphids, mites, nematodes, and weevils, further complicating pest management in banana cultivation. Plant-parasitic nematodes and weevils pose a significant global threat to banana cultivation, resulting in severe yield losses varying from 40% to 50% (Gold et al., 2001). Various nematodes, such as Radopholus similis, Pratylenchus goodeyi, Pratylenchus coffeae, Helicotylenchus multicinctus, and Meloidogyne spp., are prevalent either alone or in combination in banana fields (Coyne et al., 2013). Similarly, banana weevils (Cosmopolites sordidus) stand out as the most challenging insect pest on a global scale, inflicting severe damage to both roots and pseudostems (Twesigye et al., 2018). The escalating impact of these pests underscores the urgent need for sustainable and accessible pest management strategies.

Moreover, abiotic factors such as soil erosion, nutrient deficiencies, and climatic variability pose additional challenges to banana production. The susceptibility of banana to climatic conditions, coupled with the looming threat of climate change, further exacerbates production challenges, potentially altering traditional growing regions and exposing banana to new risks.

Addressing these multifaceted production constraints requires a holistic approach encompassing improved agricultural practices, disease-resistant varieties, sustainable soil management, and strategies to enhance genetic diversity. By adopting such a comprehensive approach, the banana industry can mitigate risks, enhance productivity, and ensure the long-term sustainability of banana cultivation.

Edible banana were originated through the natural hybridization of two wild progenitors, Musa acuminata (AA genome) and Musa balbisiana (BB genome), boast a rich tapestry of genetic diversity. This diversity is reflected in the multitude of cultivars, which are classified into various genome groups based on their genetic makeup. Among these groups are diploid banana genomes, denoted as AA or AB, and seedless triploid genomes, encompassing AAB, AAA, and ABB variations (Hinge et al., 2022). The global count of banana cultivars ranges from an estimated 300 to 1,200, showcasing the wide array of genetic variations that have emerged through natural processes and human interventions (Ploetz et al., 2007; Thierry, 2020). Diploid seeded banana are about 290 cultivars grown in Southeast Asian countries, and edible seedless triploid banana are about 650 cultivars grown worldwide (Thierry, 2020).

The importance of genetic diversity in banana cannot be overstated, particularly in the context of adapting to environmental stresses. A diverse genetic pool equips banana plants with the resilience needed to navigate through challenges posed by biotic and abiotic factors. In contrast, a lack of genetic diversity renders banana crops vulnerable to extinction, especially in the face of rapidly changing environmental conditions. Within individual genomic groups, banana exhibit varying degrees of genetic diversity, shaped by mutations and decades of selective breeding efforts aimed at enhancing desirable traits (Thierry, 2020). Diversity in plant genetic resources allows plant breeders to develop new and improved banana cultivars with desirable characteristics, including farmer-preferred traits and disease-resistance high-yielding varieties (Govindaraj et al., 2015).

Despite the inherent challenges in preserving genetic diversity within genomic groups, it remains a crucial endeavor for the long-term sustainability of banana cultivation. The conservation of diverse genetic resources serves as the foundation for breeding programs aimed at developing new banana cultivars with enhanced traits, including resistance to diseases and pests, improved yield, and better adaptation to changing environmental conditions.

The domestication of banana spans over a millennium, resulting in the emergence of numerous parthenocarpic varieties. These varieties, characterized by the absence of seeds and developed through natural hybridization, have been propagated vegetatively by farmers over generations. However, it's noteworthy that the initial domestication process likely tapped into only a fraction of the available genetic diversity present in wild banana species (De Langhe et al., 2009). Understanding the genetic diversity of Musa species is not only crucial for the preservation of biodiversity but also for addressing future food security challenges (Ortiz, 1997). The advent of CRISPR technology represents a significant leap forward in genetic manipulation, offering unprecedented precision and speed in breeding efforts. This revolutionary tool has the potential to expedite the breeding cycles of banana and facilitate the development of cultivars with tailored traits, thereby contributing to the resilience and sustainability of banana cultivation in the face of evolving environmental and agricultural landscapes.

The field of gene editing, encompassing technologies that enable precise alterations to an organism’s DNA, has witnessed significant advancements. These tools empower scientists to add, remove, or modify genetic material at specific genomic locations with unparalleled accuracy. Among the various gene editing tools, clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) systems have emerged as a cornerstone of genetic manipulation due to their speed, cost-effectiveness, precision, and efficiency, surpassing previous techniques like meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) (Tripathi et al., 2022; Ntui et al., 2023).

CRISPR/Cas9, derived from a naturally occurring bacterial immune defense mechanism against viruses, functions by leveraging RNA segments generated from CRISPR arrays to identify and bind to specific DNA sequences (Koonin and Makarova, 2009). Upon binding, the Cas9 enzyme cleaves the DNA at precise locations, initiating the process of editing. Scientists have harnessed this bacterial defense system to edit DNA by designing short guide RNA (gRNA) sequences that guide the Cas9 enzyme to target DNA sequences in cells. Once the desired DNA sequence is identified, Cas9 cuts the DNA, allowing for the deletion, addition, or substitution of nucleotides, thereby altering the genomic DNA of cells.

The CRISPR/Cas9 system encompasses two classes (Class 1 and Class 2), six types (I to VI), and several subtypes, each with distinct characteristics (Xu and Li, 2020). The CRISPR/Cas9 technology primarily comprises two essential components: the Cas9 nuclease and the gRNA. The gRNA guides the Cas9 enzyme to induce precise double-stranded breaks (DSB) at target sites in the DNA. Moreover, it detects the protospacer adjacent motif (PAM), a three nucleotides sequence, and initiates editing upstream. Subsequently, the cell’s endogenous repair mechanisms, namely non-homologous end joining (NHEJ) and homology-directed repair (HDR), come into play to repair the DNA damage. The NHEJ pathway, an error-prone mechanism, leads to random insertions or deletions (indels) at the cleavage sites, resulting in frameshift mutations and targeted gene knockouts. On the other hand, the HDR pathway enables precise genomic alterations, such as gene knock-in, gene replacement, or insertion of foreign genes or DNA sequences, by employing a homologous DNA repair template. Furthermore, the type of repair determines the classification of editing into three categories: SDN1, SDN2, or SDN3 (Modrzejewski et al., 2019). SDN1 involves random mutations in the host genome, altering gene function or causing gene silencing or knockout. SDN2 utilizes a repair template matching the DSB, leading to nucleotide substitution or targeted indels via HDR. SDN3 facilitates the targeted insertion of foreign genes by repairing the DSB with a longer repair template than the homologous sequences.

Since the advent of CRISPR technology, several CRISPR-based tools have been developed, with broadened targeting ranges, enhanced editing specificity and efficiency, and other unique functionalities, revolutionizing crop engineering (Figure 1). CRISPR/Cas9 remains the most widely utilized system due to its stability, adaptability, ease of design, and capacity to multiplex gene editing Originating from the type II CRISPR immune system in bacteria, CRISPR/Cas9 comprises the Cas9 endonuclease from Streptococcus pyogenes and a synthetic single guide RNA (sgRNA). The sgRNA directs Cas9 to a specific DNA sequence, guided by the protospacer adjacent motif (PAM), with Cas9 demonstrating a higher affinity for NGG compared to NAG. This system enables precise gene editing and simultaneous modification of multiple genes (Tripathi et al., 2023).

In addition to Cas9, several other Cas variants, including Cas12a, Cas13, Cas14 and CasX have been developed. Cas12a, also known as Cpf1, is a type V, class 2 CRISPR that only harbors the RuvC domain. It possesses RNase activity for crRNA synthesis and DNase activity for single strands. Cas12a recognition of PAM sequences like TTN/TTTN/TTTV and T-rich motifs allows for efficient multiplex gene modifications using a single sequence array on the selected gRNA (Tripathi et al., 2022). Cas13a, a class 2 type VI-A ribonuclease, targets and cleaves single-stranded RNA, offering higher accuracy in viral detection compared to traditional methods. PAM fragments are not necessary for Cas13a activity.

Cas14, an RNA-guided nuclease, uniquely recognizes DNA without PAM dependency, displaying versatile ssDNA cleavage capabilities and high specificity for single-nucleotide polymorphisms (SNPs) (Wu et al., 2022; Zhou et al., 2023). In contrast to Cas12a, Cas14 recognises SNPs with great specificity and sensitivity, which has been used for pathogen discrimination and genotyping. This characteristic has been harnessed for pathogen discrimination and environmental monitoring applications. Indirect sensing of non-nucleic acid targets using Cas14 has been investigated, allowing for the sensitive detection of antibiotics with low nanomolar sensitivity. LC-MS and the usage of metal isotopes, however, made it less straightforward. Wu et al. (2022) created a CRISPR/Cas14-based aptasensor that achieved excellent sensitivity in environmental monitoring by detecting microcystin-LR with nanomaterials-assisted fluorescence generation. However, the use of complementary DNA to block the aptamer’s binding capacity may result in signal loss in target recognition. Furthermore, the potential of CRISPR/Cas14 for aptasensing has never been investigated. Thus, it is worthwhile to try to create an affordable, quick, and direct Cas14-based biosensor for flexible aptasensing.

CasX, identified through metagenomic analysis of groundwater-derived bacteria, represents another promising variant. It functions as an RNA-guided DNA nuclease with a distinct PAM recognition pattern (5′-TTCN) (Liu et al., 2019). CasX is smaller than Cas12, with a unique structure that includes a RuvC domain. Its features include PAM-independent ssDNA trans-cleavage activity, making it a versatile tool for genetic modification. Studies have shown CasX’s potential in gene editing across different organisms, including human cells (Yang and Patel, 2019).

Recently, a new family of RNA-guided endonucleases was found that shares a core domain with the CRISPR/Cas12 family. Evolutionarily conserved, RNA-guided DNA endonucleases carry out a variety of functions in vivo, ranging from the prokaryotic TnpB effector to the eukaryotic Fanzor effector. The transposable elements TnpB and Fanzor effectors, together referred to as the OMEGA system, include a CRISPR/Cas9 like domain (RuvC) that functions as an RNA-guided endonuclease (Karvelis et al., 2021). Using uRNA that is complementary to the target DNA, TnpB helps the TnpA module to facilitate the transposition of a particular locus. This RNA guidance allows for the reprogramming of DNA targeting, which is widely employed in gene editing. It is believed that the CRISPR/Cas12 system present in prokaryotes arose from TnpB by introducing extra domains, given that these TnpBs only have a minimal core domain that performs the CRISPR/Cas12 family’s function. From the first classified ISDra2 TnpB, K, and racemifer TnpB types to the most recent database-screened ISDge10, ISAam1, and ISYmu1, the features of target DNA recognition of TnpB have been reported (Altae-Tran et al., 2021; Sasnauskas et al., 2023; Xiang et al., 2023; Badon et al., 2024). Fanzor effectors are mostly found in plants, fungi, protists, arthropods, and eukaryotic viruses. At the molecular level, they exhibit a great deal of similarities to the TnpB system. Fanzor is mainly divided into Fanzor 1 and 2 types. It has been observed that both forms use TAM sequence recognition and uRNA complementary to target DNA, much like TnpB, to produce RNA-DNA heteroduplexes on target DNA. Like TnpB and Fanzor endonucleases, IscB recognises TAM and has a small size of 496 aa (OgeuIscB). Nonetheless, it has comparable functionality, nucleic acid binding, and domain organisation (RuvC, BH, and HNH domains) (Badon et al., 2024).

Prime and base editing technologies represent significant advancements in CRISPR-based gene editing. These editing methods make use of dead Cas9 (dCas9), a modified version of Cas9. To produce a base editor that allows base substitution at single nucleotide resolution without the need for a DNA donor template, a DNA deaminase is coupled to the dCas9 in base editing. The effectors permit C:G-to-T:A or A:T-to-G:C substitution, depending on the kind of DNA deaminase, and the RNA-guided CRISPR system locates the targeted locus in the genome that has to be altered. Prime editing mediates DNA base pair swaps, minor insertions, and tiny deletions (indels) by a process similar to that of classical CRISPR/Cas systems (Matsoukas, 2020; Chen et al., 2021). In contrast, primer editing doesn’t require a donor template or result in DSB; instead, it removes off-target effects and fixes frameshifts brought on by indels. The genome can only be altered by a fusion protein made up of a longer-than-usual gRNA called pegRNA and Cas9 H840A nickase linked to a modified reverse transcriptase (RT) enzyme. Prime and base editing are SDN1 types of editing because they don’t need a DNA donor template. This suggests that they may be treated similarly to non-transgenic crops and may not be subject to stringent biosafety regulations.

These advanced CRISPR technologies offer tremendous potential for improving crop resilience, enhancing nutritional quality, and addressing global food security challenges.

Banana is one of the most consumed fruits globally playing a crucial role in food security. However, challenges such as pests, diseases, and nutritional deficiencies pose significant threats to banana production and sustainability. To address these challenges, researchers worldwide are harnessing the power of CRISPR/Cas technology to develop resilient, high-yielding, and nutrient-enriched banana varieties (Table 1).

The pursuit of transgene-free gene editing in banana aims to create non-GMO plants with desired traits while addressing regulatory constraints associated with GMOs. Despite the broader array of transformation approaches available, achieving transgene-free plants, especially in clonally propagated crops like banana, remains a challenge.

Currently, gene editing in banana involves plasmid delivery, where plasmids containing the Cas9 protein, selection marker genes, promoters, and terminators are introduced into plant cells via Agrobacterium-mediated transformation. These gene sequences integrate into the banana genome. Given that banana is vegetatively propagated crop, segregating out these sequences through crossing is impractical. Consequently, gene-edited banana is classified as GMOs by regulatory bodies and are subject to stringent biosafety regulations, which can hinder commercialization and acceptance (Tripathi et al., 2019c). To address this regulatory hazel and increase the commercialization of gene edited banana, it is imperative to produce DNA-free products. Several strategies are being explored to produce transgene-free gene-edited banana plants.

One approach involves utilizing ribonucleoproteins (RNPs), where a preassembled complex of Cas9 protein and gRNA is delivered into the plant cell (Liang et al., 2017). This complex facilitates gene editing at target sites immediately after transfection and is rapidly degraded by endogenous proteases, minimizing off-target effects and preventing the integration of foreign DNA elements (Woo et al., 2015). Various delivery methods such as electroporation, particle bombardment, and protoplast transfection have been explored for direct delivery of the RNA-guided engineered nucleases- ribonucleoproteins (RGENs-RNPs) into plant cells, with protoplast transfection being the most versatile. While some authors have reported the regeneration of complete plants from banana protoplast (Panis et al., 1993; Matsumoto and Oks, 1998; Assani et al., 2001), regenerating plants from banana protoplasts remains challenging (Tripathi et al., 2022).

Another strategy involves transiently delivering the editing machinery into plant cells via Agrobacterium without applying selection. This method has been demonstrated in other crops, resulting in the production of transgene-free plants. For example, Chen et al. (2018) produced transgene-free tobacco plants by transient expression of CRISPR/Cas9 containing gRNAs targeting the PDS gene. They obtained up to 8.2% non-transgenic mutants. Using a similar approach, Veillet et al. (2019) modified the acetolactate synthase (ALS) gene via a cytidine-based editor and obtained transgene-free potato and tomato plants with mutation efficiency of 10% and 12.9%, respectively. However, challenges such as high off-target effects and the need for extensive screening remain.

To address these challenges, researchers are designing plasmids with mechanisms for T-DNA excision and removal following editing (Dalla Costa et al., 2020). Techniques such as the Flp/FRT system and synthetic cleavage target sites (CTS) have been developed to remove T-DNA from CRISPR-edited plants. Although challenges with trimming at T-DNA boundaries exist, these approaches represent significant progress toward producing transgene-free plants.

In ongoing research, efforts are underway to refine the process of producing transgene-free banana through transient delivery of the Cas9-gRNA reagent by Agrobacterium. Additionally, procedures for regeneration, PEG transfection, and protoplast isolation are being developed to streamline the process.

While transferring CRISPR/Cas9 plasmids into germ lines or protoplasts presents technical challenges and inefficiencies, innovative approaches such as de novo induction of meristems offer promising avenues for overcoming these limitations in dicotyledonous plants. This approach involves delivering developmental regulators and gene-editing components into somatic cells of entire plants, resulting in the transmission of desired DNA modifications to the next-generation. The graft-mobile gene editing system can be another strategy to the production of transgene-free plants in one generation without the need for transgene segregation (Yang et al., 2023).

Gene editing is predicted to usher in a new Green Revolution, enhancing food and nutritional security worldwide and mitigating the effects of climate change. However, the technology also raises significant ethical concerns that encompass environmental, social, and economic dimensions.

One major concern is the possibility of off-target effects, edits occurring in unintended locations, which can result in unwanted phenotypes. The aspect of safety has been one of the critical issues of gene editing. There are questioned surrounding the safety of gene editing, especially when the technology is used for gene drives, since off-target effects in gene editing are not fully understood. There is also concern that gene-edited crops could reduce biodiversity if they outcompete natural species or if large-scale monoculture practices are adopted.

Another significant issue is the regulatory framework for gene editing. There is ongoing debate over whether gene-edited crops should be categorized as GMOs (Karalis et al., 2020). While some countries have clarified their regulations regarding gene edited crops, others, such as the European Union, South Africa and New Zealand, maintain very strict regulations, leading to variations in national regulatory strategies. Policy and regulation development for gene editing in plant breeding must also consider factors like farmers' rights and public acceptance (Idris et al., 2023). As with many new technologies, there is concern of Intellectual property rights (IPR). Gene-edited products will inevitably be patented, providing owners with IPR rights—typically agri-food corporations—with what amounts to monopolistic control over the gene-editing process' output (Sprink et al., 2022). The patenting of gene-edited crops by corporations can lead to concerns about farmers' rights and their dependency on a few large companies for seeds, potentially driving up costs and limiting traditional farming practices.

Moral and religious objections also play a significant role in the ethical debate. Many people believe that gene editing interferes with natural creation, equating it to “playing God.” These objections highlight the need for inclusive and culturally sensitive discussions when considering the widespread adoption of gene editing in agriculture.

CRISPR/Cas based gene editing stands as a transformative technology with vast potential for enhancing crop productivity and nutritional quality, thus bolstering global food security amidst mounting environmental challenges. However, the clonal propagation of banana presents unique challenges in integrating gene-edited traits due to the seedless nature of the fruit. While plasmid-based delivery systems and embryogenic cell methods offer feasible pathways for generating gene-edited banana plants, the task of segregating transgenes through conventional breeding is hindered by the lack of seeds in banana. Overcoming these hurdles requires optimization of techniques like using RNPs for generating plants from protoplasts or microprojectile bombardment of cell suspension. Additionally, robust protoplast regeneration systems must be developed through further research to facilitate the creation of transgene-free plants in banana cultivars with shorter breeding cycles. Disease resistance trait is successfully targeted by knocking off the susceptible genes in the banana genome, like DMR6 and Early Nodulin gene. However, challenges persist in targeting complex polygenic traits like abiotic stress tolerance, necessitating the simultaneous knockout of multiple genes or targets.

Nevertheless, the diligent exploration of innovative technologies such as CRISPR/Cas holds promise for delivering high-yielding better banana with enhanced nutritional content and disease resistance. By integrating precision genetics with traditional breeding programs and adopting transgene-free strategies, researchers can unlock the full potential of gene-edited banana. The future holds exciting prospects for the development of banana that not only thrive in diverse environments but also offer superior nutritional value, benefiting farmers and consumers alike.