The cultivated strawberry (Fragaria ×ananassa) is among the most economically important fruit crops in the world. According to the Food and Agricultural Organization of the United Nations, during 2021, more than nine million tonnes of strawberries were produced from 389,665 ha around the world (FAO, 2023). The cultivated strawberry as it is today resulted from a series of interspecific hybridizations, with the final cross between octoploid progenitors occurring only approximately 300 years ago (Edger et al., 2019; Whitaker et al., 2020). Evolutionary analysis of Fragaria ×ananassa suggested genomic contributions of four diploid progenitor species: Fragaria vesca, Fragaria iinumae, Fragaria viridis, and Fragaria nipponica (Edger et al., 2019; 2020). However, analysis from other groups has suggested that both F. viridis and F. nipponica are not among the diploid progenitors (Liston et al., 2020; Jin et al., 2023; Lyu et al., 2023; Session and Rokhsar, 2023). As identification of the progenitor species may enable greater prediction of polyploid responses to environmental stress and climate change (Liston et al., 2020), further analysis of the evolutionary history of strawberry will be necessary to confirm the identities of the diploid progenitor species.

Fragaria ×ananassa is a highly heterozygous, allo-octoploid species (2n = 8x = 56) with a phased genome length of approximately 780 Mb (Hardigan et al., 2021a; Han et al., 2022). Due to the complex nature of the allo-octoploid genome and high genomic heterozygosity, trait discovery and gene functionalization studies in strawberry are commonly performed in the diploid woodland strawberry, Fragaria vesca. Fragaria vesca is used as a model system for strawberry research for several reasons: it has a small genome size of approximately 240Mb, has a short life cycle, is relatively responsive to transformation, and is easy to propagate using both seeds and runner cuttings (Oosumi et al., 2006). Fragaria vesca is additionally the closest relative of the transcriptionally dominant diploid subgenome of Fragaria ×ananassa (Hardigan et al., 2021b), further lending to its value as a model system for strawberry research. While trait discovery and gene functional studies performed in Fragaria vesca are useful for inferring mechanisms of commercially valuable traits in the cultivated strawberry, there are also drawbacks to relying on the diploid system. The largest drawback to working with the diploid strawberry is that Fragaria vesca tends to be more homozygous at a given locus as compared to Fragaria ×ananassa. Martín-Pizarro et al. (2019) identified one such instance of this when they identified at least five alleles of the FaTM6 locus across the four homoeologous chromosomes of octoploid strawberry and only a single homozygous FvTM6 locus in the diploid strawberry. Due to the differences in heterozygosity, it can be difficult to determine which allele(s) in Fragaria ×ananassa contribute to a phenotype using data from Fragaria vesca alone, hindering attempts at genomics-assisted improvement of commercially relevant varieties.

Recent advancements in genome sequencing technology and gene annotation have enabled the assembly of numerous strawberry genomes encompassing a range of ploidy levels (Qiao et al., 2021; Han et al., 2022; Song et al., 2023a). Enabled by these genome assemblies, CRISPR/Cas genome editing is increasingly applied for the study and improvement of key strawberry traits. While not currently widespread due to several challenges, genome editing in allo-octoploid strawberry offers a unique opportunity to precisely modify traits of interest. Alternatively, tools such as RNAi, stable or transient overexpression of transgenes have been utilized to validate gene function and support identification of candidate genes. Plant breeding has also made use of genomics-assisted techniques such as marker-assisted selection and genomic selection for varietal improvement, enabled by new methods for QTL discovery and analysis.

This review covers the genomic tools and technologies available for genomics-assisted improvement of cultivated strawberries, in addition to multi-omics technologies and their applications in identifying candidate QTLs and genes. Reports of successful CRISPR/Cas genome editing for traits associated with strawberry fruit quality and production are discussed herein. In addition to these reports, this review suggests additional candidates for genome editing based on analyses in strawberry and other crops.

Genomic complexity of the polyploid genome is one of the major challenges of genome editing in octoploid strawberry. The first high quality, chromosome-scale reference genome of Fragaria ×ananassa cv. ‘Camarosa’ was developed in 2019 ​(Edger et al., 2019)​. Before the release of the first octoploid strawberry genome, there were few genome assemblies available for polyploid species, as the homoeologous nature of the subgenomes made assembly of sequences difficult (Wang et al., 2023b). As such, molecular genetic analysis of Fragaria ×ananassa prior to 2019 relied on early genome assemblies of Fragaria vesca, the first of which was published in 2011 (Shulaev et al., 2011). Recent rapid advancements in genome sequencing technology have resulted in the release of additional annotated octoploid genome sequences of increasing quality (Table 1) (Wang et al., 2023b). Advancements in genome sequencing in strawberry have not been limited only to the octoploid, as new high-quality genomes have also recently been released for the diploid model species, Fragaria vesca (Alger et al., 2021; Joldersma et al., 2022). As improvements to genome sequencing technology continue, it will be possible to better understand genomic complexity in not only octoploid strawberry, but in other polyploids as well.

Recently, multiple single nucleotide polymorphism (SNP) arrays for octoploid strawberry have been developed to assist with quantitative trait loci (QTL) discovery, including 50K and 90K arrays (Bassil et al., 2015; Verma et al., 2017). Using these tools to identify SNPs correlated to QTLs enables the development of molecular markers which can be utilized in breeding (Jung et al., 2017). Marker assisted selection (MAS) is a method of precision genomics-assisted breeding which relies on the implementation of genetic markers and trait associations to inform selection (Collard and Mackill, 2008). Large numbers of marker-trait associations have been generated through QTL mapping studies (Collard and Mackill, 2008; Rey-Serra et al., 2021) which makes MAS a powerful tool for precision breeding. Implementation of high-throughput assays such as high-resolution melting (HRM) and simple sequence repeat analysis (SSR) paired with DNA markers and rapid high-throughput DNA extraction methods have enabled rapid improvement of octoploid strawberry varieties at the University of Florida (Noh et al., 2017). This success highlights the significant potential of MAS for varietal improvement of fruit crops.

Genomic selection (GS) is a method of selection which utilizes genome-wide variation and phenotypic data to predict phenotypes of an unobserved population (Bernardo, 1994; Meuwissen et al., 2001; Goddard and Hayes, 2007; Montesinos-López et al., 2021). GS offers the potential for increased genetic gain within a breeding program, as it enables increased selection intensity, selection accuracy, and reduction of the generational interval (Werner et al., 2023). Additionally, GS enables faster selection of clonally propagated crops such as those in the Rosaceae family by predicting their performance as clones while they are still in the seedling stage, allowing for earlier analysis of traits that would otherwise require further physiological development, such as fruit flavor and shelf life (Werner et al., 2023). Due to the complexity of the polyploid genome, generation of genome-wide SNP arrays can be complicated, contributing to the delay in adoption of GS methods for breeding of polyploid crops (Zingaretti et al., 2019). Despite the challenges posed by genomic complexity of polyploids, the continuous release of high-quality reference genomes has allowed for the parallel improvement of SNP genotyping methods. To aid in adoption of GS in polyploids, software such as polyploid Sequence Based Virtual Breeding (pSBVB) has been developed to simulate and evaluate GS strategies in polyploids and is equipped to simulate differences between allo- and autopolyploids (Zingaretti et al., 2019). Numerous reports have now been made for successful application of GS in octoploid strawberry (Gezan et al., 2017; Whitaker et al., 2017; Osorio et al., 2021). In a study by Yamamoto et al. (2021), 105 inbred strawberry lines were developed and used to train a GS model based on phenotyping data for petiole length, leaf area, Brix, fruit firmness, and pericarp color. Using the model to predict phenotypic values for a F1 hybrid testing population derived from these 105 lines revealed that phenotypic data collected from the parental inbred lines was sufficient to predict the F1 hybrid phenotypes when the model accuracy in cross-validation is sufficient (Yamamoto et al., 2021). Pincot et al. (2020) applied GS to strawberry to evaluate improve Verticillium wilt resistance. While the inclusion of wild genotypes in the training population reduced accuracy, the results suggested a strong potential for GS to identify superior resistant individuals if the model was sufficiently trained (Pincot et al., 2020).

Due to the wide range of research in strawberry, the volume of available genomic resources has increased significantly. As such, the Genome Database for Rosaceae (GDR) was created to house a wide range of tools and data. Among these tools is a compendium of strawberry DNA tests which can be downloaded from GDR ¹ and implemented in breeding programs (Oh et al., 2019).

Following trait discovery, genetic transformation can be performed to validate candidate gene function (Figure 1). While CRISPR/Cas-mediated genome editing has yet to be widely implemented and optimized for Fragaria species, numerous reports of transgenic modifications have been made. Agrobacterium-mediated transformation is currently the most applied transformation method for strawberry. Protocols using agrobacterium have undergone significant optimization to improve transformation efficiency. Thus far, protocols have been established for Agrobacterium-mediated transformation and subsequent regeneration of a range of tissues (Li et al., 2018a; Feng et al., 2019; Feng et al., 2021b; Wilson et al., 2019; Duan et al., 2021; Mao et al., 2022; Yan et al., 2023). In strawberry, agrobacterium-mediated transformation most commonly uses Agrobacterium tumefaciens strains LBA4404, GV3101, and MP90. Leaves are the most common explant material (Table 3). Transient methods of agrobacterium-mediated transformation have also been developed for fruit (Carvalho et al., 2016; Dai et al., 2020; Zeng et al., 2021; Mao et al., 2022; Lee et al., 2023) to study a range of mechanisms and traits, as well as to analyze the performance of DNA constructs prior to stable transformation. Particle bombardment (Agius et al., 2005) and protoplast transfection (Gou et al., 2020) have also been applied in strawberry for transient analyses. However, despite the demonstrated success of both methods in such analyses, stable transformation of strawberry with these approaches remains a challenge.

Transformation of strawberry has also been performed using the hairy root system (Yan et al., 2023). Hairy roots are valuable for functional analysis of root traits as well as for validation of transgenic and genome editing methods due to the relatively short period of root development ​​(Ozyigit et al., 2013)​. Several advancements have been made to Agrobacterium-mediated transformation technologies, including improved ternary systems (Anand et al., 2018), auxotrophy of various amino acids (Aliu et al., 2020; Prías-Blanco et al., 2022), and use of a CRISPR RNA-guided integrase system (Vo et al., 2021; Aliu et al., 2022), however, there has been little application of these advanced systems in strawberry (Oosumi et al., 2006).

Some transgene-free methods, such as ribonucleoprotein (RNP) bombardment and RNP transfection of protoplasts, have not been reported in strawberry but have demonstrated success in a range of crops (Liu et al., 2020; Zhang et al., 2021a; Najafi et al., 2023). Other methods of transformation and editing, such as RNA bombardment (Li et al., 2019a) and virus-mediated transformation (Tian et al., 2015) have been sporadically applied in strawberry, indicating a need for further development. Transgene-free targeted mutagenesis approaches for varietal development fall within improved consumer acceptance and reduced regulatory constraints. Thus, continued refinements of genomic tools, transformation and genome editing strategies will position CRISPR/Cas technology as primary tool for gene function validation and crop improvement.

Further development of efficient protoplast isolation, transformation, and regeneration is also important for future transgene-free editing of strawberry. Isolation and regeneration of protoplasts is well established for strawberry (Nyman and Wallin, 1988; 1992a; Barceló et al., 2019; Gou et al., 2020). However, while protocols have been established for transient analyses in protoplasts, few reports of successful regeneration involve transformed materials (Nyman and Wallin, 1992b; Pattanaik et al., 2004; Gou et al., 2020). As such, it is necessary to continue developing methods to transform and regenerate plants from strawberry protoplasts as a foundation for transgene-free genome editing.

As has been established in other species, transformation and regeneration of strawberry depends on several factors, and protocol optimization can be challenging. Experiments to optimize strawberry transformation have observed a wide range of transformation and regeneration efficiencies which vary significantly between genotypes (Zakaria et al., 2014). Other factors, such as transformation method and explant material, have also been reported to impact regeneration efficiency, and response to these factors also varies strongly by genotype (Table 3). Taken together, these findings suggest that some genotype-specific optimization of protocols will be necessary for efficient genetic transformation. Additionally, transformation and regeneration efficiencies tend to be higher for diploid strawberry than octoploid strawberry, even when other factors are held constant between species (Table 3). As Fragaria ×ananassa is the species of economic interest, continued optimization to improve both transformation and regeneration efficiencies will be essential to facilitate genome editing for varietal improvement.

CRISPR, or Clustered Regularly Interspaced Palindromic Repeats, is a genome editing system derived from a bacterial defense network. In bacteria, this defense network operates in two phases to incorporate short fragments of invading DNA into the bacterial genome and then use these sequences to recognize and cleave foreign DNA based on the presence of a protospacer adjacent motif (PAM) (Doudna and Charpentier, 2014; Vigouroux and Bikard, 2020). For genome editing via CRISPR/Cas systems, this bacterial defense pathway is manipulated to target specific sequences within a genome of interest. Unlike other methods of genome editing, such as zinc-finger nucleases (ZFNs) and transcription activator-like nucleases (TALENs), which require substantial protein engineering, CRSIPR/Cas genome editing can be performed simply through a change in the single guide RNA (sgRNA) sequence (Doudna and Charpentier, 2014). The ability to switch editing targets quickly without need for protein engineering has played a major role in the rise of popularity of CRISPR/Cas genome engineering. Double stranded breaks (DSBs) are generated when a Cas endonuclease cleaves DNA at a targeted site using a sgRNA as reference (Cong et al., 2013; Mali et al., 2013; Doudna and Charpentier, 2014). These DSBs can then be repaired through non-homologous end joining (NHEJ), or template mediated homology-directed repair (HDR). NHEJ is error prone, often resulting in insertions or deletions that cause loss of gene function (Cong et al., 2013; Mali et al., 2013; Chen et al., 2022). Targeted mutagenesis using NHEJ mediated repair of CRISPR/Cas-mediated DSBs has been reported in many Rosaceous crops, including apple (Malnoy et al., 2016; Pompili et al., 2020), pear (Charrier et al., 2019; Pang et al., 2019), raspberry (Miller, 2019), and strawberry (Martín-Pizarro et al., 2021; López-Casado et al., 2023). In contrast, template mediated HDR allows precise conversion of targeted single nucleotides or insertion of a specific sequence. Allelic variants differing in single-nucleotide polymorphisms often confer improvement of agronomic traits. HDR pathways can be leveraged to replace alleles with superior variants and has successfully been implemented in crops such as maize, rice, and sugarcane (Shi et al., 2017; Wang et al., 2017; Oz et al., 2021). Reports of HDR-mediated gene targeting are still lacking in strawberry, likely due to low efficiency caused by infrequent occurrence of HDR, competition with NHEJ for DSB repair, and inadequate repair template in close proximity to the DSB site (Chen et al., 2022).

Similar to template mediated HDR, both base and prime editing can generate precision nucleotide substitutions in target genes. Base editing occurs as the result of a catalytically impaired Cas nuclease, such as Cas nickase (nCas) or dead Cas (dCas), fused to a nucleotide deaminase (Molla et al., 2021) and results in an irreversible base conversion without the need for DSBs or an exogenous template (Azameti and Dauda, 2021). Base editing requires the use of different deaminases depending on the desired nucleotide substitution, is currently limited to six of the 12 possible base-swaps, and may result in bystander mutations (Molla et al., 2021). In contrast, prime editing, which occurs as the result of fusing a nCas nuclease with a reverse transcriptase, is capable of generating all 12 possible substitutions as well as small indels in exchange for lower editing efficiency (Molla et al., 2021). Base editing has been applied for the creation of precision nucleotide substitutions in strawberry to support the fine tuning of the sugar content of the strawberry fruit (Xing et al., 2020). While prime editing has not been reported in strawberry, it has been successfully applied in tomato, rice, and wheat (Lin et al., 2020; Xing et al., 2023), demonstrating its potential for precision nucleotide substitution in plant systems.

In addition to generating nucleotide substitutions through base or prime editing, CRISPR/Cas can also be applied to modulate gene expression patterns and epigenetic regulation. By fusing dCas with different effector proteins, it is possible to achieve efficient targeted activation (CRISPRa), repression (CRISPRi), or epigenome modifications (Pan et al., 2021).

While genome editing using the CRISPR/Cas system has largely focused on the use of the Cas9 endonuclease, additional Cas nucleases have been engineered to improve the flexibility of the CRISPR genome editing system by relaxing the requirements for a specific protospacer adjacent motif and altering nuclease function (Guilinger et al., 2014; Shen et al., 2014; Trevino and Zhang, 2014; Tsai et al., 2014; Anders et al., 2016; Komor et al., 2016; 2017; Havlicek et al., 2017; Harrington et al., 2018; Liu et al., 2019; Anzalone et al., 2020; Ghogare et al., 2020; Zhang et al., 2021b; Sukegawa et al., 2023). This is particularly useful for generation of precision nucleotide substitutions using base or prime editing (Wang et al., 2020b; Kantor et al., 2020; Mishra et al., 2020; Huang and Puchta, 2021).

Genome editing is a powerful tool for crop improvement, as it allows for precise, targeted mutation in one or few genes without altering the plant’s genetic background. Genome editing can be an efficient method for varietal improvement as co-editing of multiple genes or alleles by multiplex editing allows researchers to accelerate the generation of desired combinations in elite germplasm without undergoing meiotic recombination (El-Mounadi et al., 2020). In contrast, conventional breeding schemes typically require numerous generations and backcrossing to improve gene or allele combinations for a single trait of interest. Genome editing with CRISPR/Cas also enables the introduction of traits that do not exist within a breeding germplasm. For example, there are currently no widely available breeding sources of resistance to Botrytis cinerea in strawberry, and previous breeding efforts to increase resistance to Botrytis fruit rot (BFR) have been ineffective (Petrasch et al., 2019). However, substantial research has been performed to identify susceptibility genes related to BFR and other strawberry diseases which could make for useful knockout targets in the future.

As with other crops, commercial release of genome edited strawberry will depend on the regulatory frameworks established by individual countries or regions. Starting with the release of the framework 7 CFR Part340 ² in 1987, genome edited and other genetically plants in the United States were regulated as part of a coordinated framework which includes the U.S Department of Agriculture Animal and Plant Health Inspection Service (USDA-APHIS), the U.S Environmental Protection Agency (EPA), and the U.S Food and Drug Administration (FDA) (United States Department of Agriculture, 2022; United States Food and Drug Administration, 2024). Seven CFR Part 340 underwent revisions in 2020, which included several updates to the existing regulatory system based on 3 decades of research and experience (United States Department of Agriculture, 2022). These updates provide a better breakdown of eligibility for non-regulated status. Application of CRISPR/Cas genome editing strategies can meet multiple of these eligibilities by introducing targeted single base-pair substitutions (7 CFR 340.1(b) (2)) or modifications which are present within the plant’s gene pool (7 CFR 340.1(b) (3)). In addition to the United States, several countries around the world have begun loosening restrictions on genome edited plants (Menz et al., 2020). While the European Union was slower to adopt looser regulations, the European Parliament recently voted to lessen regulatory oversight (Stokstad, 2024), representing a major step forward for global regulation of plant biotechnology.

At this time, there are no commercially available genome edited strawberry varieties. However, other edited species have undergone commercialization. In 2016, non-browning mushrooms were the first CRISPR/Cas9 genome edited organism to pass USDA regulation (Waltz, 2016). Pairwise recently released genome edited Brassica juncea with reduced pungency for improved flavor (Brown, 2023). Pairwise has also recently received nine new exemptions from USDA regulation for berry crops, bringing the company’s confirmed exemptions to 19 for berries and 21 in total (Barefoot, 2024). However, these genome edited berries have not been commercially released at the time of this review. These examples and others suggest a bright future for the de-regulation and commercialization of genome edited crops.

Cultivated strawberries are polyploid, highly heterozygous, and clonally propagated, which makes them difficult to improve through conventional methods. While application of genome editing in diploid woodland strawberry, Fragaria vesca, is helpful for validating gene function and identifying new candidate genes underlying traits of interest, the allo-octoploid cultivated strawberry, Fragaria ×ananassa, is the species of economic interest. Due to greater genomic complexity and the presence of homoeologous diploid subgenomes which are not closely related to Fragaria vesca, gene functions observed in woodland strawberry may not be completely conserved in octoploid strawberry. Thus, validation of gene function in the octoploid background is necessary for further elucidation of pathways underlying traits of interest. In turn, greater understanding of these pathways may then enable precise improvement of strawberry fruit quality and production traits through the application of genomics-assisted breeding techniques or the application of genome editing. Additionally, application of techniques such as base editing and prime editing to make precision nucleotide substitutions, or leveraging HDR to substitute alleles for superior variations, will enable additional opportunities for genomic improvement of strawberry.

While the application of genome editing for crop improvement is still relatively new to Fragaria ×ananassa and faces its own significant challenges, several examples of successful genome editing have been reported. These successful reports demonstrate the significant potential of genome editing in strawberry, highlighting the necessity to continue optimizing genome editing methods for improvement of economically important fruit traits. Based on the results of genome editing in Fragaria vesca, it may be beneficial to perform editing of orthologs of these genes in octoploid strawberry, to further confirm their roles in the commercially relevant species. Based on the results of transient analyses, some genes associated with desirable fruit traits may also be promising targets for genome editing. In the case of genes which have demonstrated negative effects on desirable traits when knocked out or transiently suppressed, further elucidation of their related pathways may reveal other associated genes which are better targets for application of genome editing and/or may permit fine-tuning of important fruit traits.