1 Introduction

Changing climate conditions are affecting crop production differently due to increased stressors such as soil salinity, drought, and the emergence of new diseases and insects [1] Salinity in the soil is one of the big environmental issues that adversely affect agricultural productivity. Stress due to salt can negatively affect plant growth and yield in a variety of ways. Among them are ionic toxicity, osmotic stress, and nutritional deficiencies [2, 3]. As a result of salinity stress, agriculture is particularly susceptible, which has already affected over 20% of agricultural land worldwide and is expected to continue spreading. Moreover, it has encompassed 954 million hectares of the Earth's surface (Hafeez et al.; Zaman et al.) [4, 5]. In terms of productivity, yield, and crop quality, soil salinity plays a crucial role in abiotic stress [6]. Plants adjust to salt stress in a variety of ways including osmotic, oxidative, and membrane homeostasis, as well as maintaining essential metabolic activities and phytohormones [7]. Additionally, salinity has been shown to lead to oxidative stress as a result of the production of reactive oxygen species (ROS) [8, 9]. As a result of decreased osmotic potential due to salt stress, the roots of plants experience increased osmotic stress and ion toxicity. The central dogma (CD) of molecular biology, which describes the flow of genetic information from DNA to RNA to protein, serves as the foundation for developing salt-tolerant plants. Furthermore, DNA replication, epigenetic changes, gene activators, transcription, posttranscriptional modifications, translation, posttranslational modifications (ubiquitin), transcription factors are the most important CD mechanisms that control genetic flow [10, 11]. Plants have strictly controlled CD mechanisms in order to preserve genetic integrity throughout the life cycle and carry genetic material to the next generation [12]. Crop development can be accelerated to feed the expanding global population by the engineering of numerous CD mechanisms involved in gene regulation [13].With the advent of new genome-editing technologies, including clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (cas9) crops have been genetically modified in an extremely targeted and precise manner, which has accelerated the trend towards precision breeding [14,15,16,17,18]. This technology enables precise genome editing, allowing for targeted changes to these key genes. For instance, genes encoding ion transporters, Osmo protectants, or stress response transcription factors can be modified to improve their function or expression under salt stress conditions. A major advantage of this approach is its ability to target one or more negative regulatory pleiotropic plant genes, which can be used to improve the traits of plants on multiple levels. Compared to prior editing technologies (Transcription activator-like effector nucleases (TALEN), Zinc-finger proteins (ZNFs) and meganucleases) CRISPR/Cas9 is more versatile, simpler, and reasonably priced, making agricultural genetic engineering feasible [19, 20]. CRISPR technology makes it feasible to modify CD processes to tweak DNA, RNA, which was previously impossible [21, 22]. Thus, scientists can manipulate specific genes and their expression pathways that are involved in salt tolerance to improve stress response. This technology allowed the development of salt tolerant varieties for agronomically important crops such as rice (Oryza sativa L.) and wheat (Triticum aestivum L.) [23,24,25].Also, success in enhancing salt stress tolerance in crops has been attained using traditional breeding, mutagenesis, and genetic modification. Traditional breeding has encompassed the selection and hybridization of salt-resistant plant varieties, leading to the creation of crops with heightened resilience. Mutagenesis has provoked genetic alterations through physical or chemical means to generate salt-tolerant mutations. Genetic modification has provided more precise remedies by inserting specific genes linked to salt tolerance, including those responsible for ion transporters, Osmo-protectants, and stress-responsive proteins, into desired crops. These strategies have collectively played a role in cultivating crops with enhanced salt stress tolerance, albeit each approach presenting its own unique hurdles and constraints. The combination of understanding genetic pathways (central dogma) and precise gene editing (CRISPR/Cas) provides powerful tools for engineering plants with increased salt tolerance, paving the way for more resilient crop varieties. However, CRISPR/Cas technology faces limitations such as off-target effects, where unintended parts of the genome are edited, potentially causing undesired mutations. Additionally, the delivery of the CRISPR/Cas components into plant cells can be challenging, and regulatory hurdles and public acceptance issues also pose significant obstacles to its widespread use in crop engineering. With these considerations in mind, this review paper provides a summary of the most recent CRISPR-based CD modulations to confer salt tolerance in crops [25].

2 Salt stress and its effect on plants at gene level

Salt stress, caused by high salinity levels in the soil or water, has a negative impact on plant growth and productivity. It causes osmotic stress, which reduces plant water uptake and leads to dehydration. High salt concentrations can also cause ionic stress, a condition in which excess sodium (Na+) and chloride (Cl−) ions accumulate in plant tissues, disrupting cellular functions and nutrient balance. These conditions impede photosynthesis, reduce leaf area, and limit overall plant growth. Additionally, salt stress causes oxidative stress, which produces harmful reactive oxygen species (ROS) that damage cellular structures. Plants may respond to stress by producing compatible solutes, ion compartmentalization, and antioxidant enzymes, but prolonged exposure can eventually lead to plant death. Currently, soil salinity has become a serious problem for plants, lowering crop yields all over the world. In the form of soluble salts, vital nutrients are absorbed by plants; however, an excessive buildup severely stunts plant growth [26]. Plants under salt stress experience both osmotic and ionic stress (Fig. 1). Plant roots immediately experienced osmotic stress after being exposed to salts, which cause cell expansion and water uptake inhibition [27]. Secondary stresses like oxidative stress, which harms membrane lipids, proteins, and nucleic acids, are frequently brought on by osmotic and ionic stress [28, 29]. Ionic stresses that result in chlorosis, necrosis, and a reduction in the activity of cellular metabolism, including photosynthesis, can be brought on by toxic ions (like Na+) building up inside of cells. The SOS (Salt Overlay Sensitive) pathway is the best understood salt signaling pathway, and excess sodium ions in this pathway increase free Ca2+concentrations in the cytosol. This increased Ca2+ level acted on a CIPK24 (Ser/Thr type CBL interacting protein kinase), and activated CIPK24/CBL4 activated membrane bound Na+/H+ antiporter and other transporters, resulting in ion homeostasis and stress resistance [30, 31]. Plant leaves are the main site of sodium ion toxicity. To prevent the accumulation of toxic Na+ ions in the leaves, roots alter ion transport, resulting in reduced long-distance transport of Na+ ions to the shoots. The key players in this process are Na+/H+ antiporters, which act primarily to efflux Na+ ions from the cell. Na+ influx in roots is mediated by nonselective cation channels, such as cyclic nucleotide-gated channels or some members of the high-affinity K+ transporter (HKT) family [32, 33] (Fig. 2).

Fig. 1
figure 1

Various effects of salt stress on the plants

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Fig. 2
figure 2

The molecular mechanisms of plants under salt stress: Salt stress is sensed by the plants at the plasma membrane. SOS (Salt Overlay Sensitive) pathway is the best-characterized pathway of salt signaling and the excessive sodium ion in this pathway induces increment free Ca2+ concentrations in the cytosol. This elevated level of Ca2+interacted with a CIPK24 (Ser/Thr type CBL interacting protein kinase) and this activated CIPK24/CBL4 activates membrane-bound Na+/H+ antiporter and other transporters resulted in ion homeostasis and stress tolerance. During osmotic stress, greater ROS synthesized that activates transcription factors which are involved in the activation of stress-responsive genes. Genes involved in detoxifying stress are ROS scavenging enzyme genes, Osmo-protection compounds synthesis genes, vacuolar sequestration genes and molecular chaperones. In vacuole also, higher salt in cell sequestrated via the NHX transporter during the salt stress. NSCCs, nonselective cation channels; ROS, reactive oxygen species; CDPKs, calcium-dependent protein kinases; CBLs, calcineurin B-like proteins; CIPKs, CBL-interacting protein kinases; AP2/ERF, APETALA2/ETHYLENE RESPONSE FACTOR; bZIP, basic leucine zipper; NHX, Na+ /H+ exchanger; SOS, SALT OVERLY SENSITIVE

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2.1 Effect on roots growth and number

Plants and soil are connected via roots and due to higher water pressure in plant roots than the soil solution, plants can absorb water. Contrary to it, when a plant is under salt stress, it cannot absorb enough water because the osmotic pressure of the soil solution becomes higher than that of the plant's cells [34]. Root system characteristics such as root length, root diameter, root number, number of root hairs, xylem vessel diameter, root cortex width, and suberin deposition are affected by salt stress [35]. Roots grown in salinity exhibit extensive vacuolization and lack the normal organization of apical tissue. Cortex and pith parenchyma cells exhibit shrinkage under salt stress. Salt stress suppresses cell division and elongation, lowers cyclin levels, and inhibits root growth [36]. According to reports, salt stress decreases the size of the root meristem, preventing primary root elongation [37,38,39]. In one such study, the researchers discovered that salt stress significantly shrinks the size of the root meristem in Arabidopsis thaliana by reducing auxin accumulation and suppressing auxin signaling by raising the levels of the nitric oxide. It is well known that auxin plays an important role in the development and growth of roots [40].

Additionally, prior studies have demonstrated that salt stress affects the growth of lateral roots, the production of root hairs, and the gravitropism of roots [41]. Some plants show extensive root growth to overcome salt stress. In a study conducted by Arif et al., [42], effect of salt stress on root morphology and root hair traits in Brassica napus was studied. In comparison to the control, the salt-treated crops had significantly longer lateral root hairs and greater root hairs. A 20% increase in estimated root surface area under salt stress conditions enhances the plants' natural water and nutrients uptake capacity that helped them to survive during difficult circumstances.

2.2 Effect on seed germination

Salt stress during seed germination is essential for the establishment of plants. Seed germination is a crucial stage in the life history of plants. Salinity has a variety of impacts on seed germination. The lower osmotic potential of the germination medium alters absorption of water, leading to toxicity that alters how nucleic acid enzyme’s function, variations in protein metabolism, upsets the hormonal balance, and hinders seeds' ability to use seed reserves [43]. During this stage, salt stress can both inhibit and prolong seed germination. The emergence of a radical through the seed coat is defined as seed germination. High salt concentrations have a detrimental effect on radicle elongation because they reduce the turgor of the radicle cells [44]. Salinity also disrupts the hormone and nutrient balances during germination, particularly gibberellin (GA) and abscisic acid (ABA). Because of this, high salinity levels may even prevent seed germination depending on the plants' tolerance to salt. It has been discovered that plant hormones, especially GA and ABA, strictly regulate germination. While GAs release dormancy and stimulate germination, ABA encourages seed dormancy and inhibits seed germination [45]. In Solanum lycopersicum, Kaveh et al. [46] discovered a significant negative correlation between salinity and the rate and percentage of germination, resulting in delayed germination and a lower germination percentage.

2.3 Effect on photosynthesis

Salinity of the soil also affects photosynthesis by reducing leaf area. With prolonged salt stress, old leaves develop chlorosis and drop. Young leaves cannot obtain the necessary carbohydrates from the plant's photosynthetic capacity if the rate of leaves senescence out paces the growth rate of new leaves and this ultimately slows down plant growth rate [36]. Photosynthesis is affected by salt stress in both the short and long term. Short-term salt stress occurs quickly and within a short period of salt exposure, affecting photosynthesis through stomatal limitations and resulting in decreased carbon assimilation [47]. Long-term salt stress has an impact on photosynthesis because salt builds up in young leaves, lowering the content of chlorophyll and carotenoids. Changes in the lipid-to-protein ratio of pigment-protein complexes or increased chlorophyllase activity could be to blame for the decrease in chlorophyll content. [36]. Under treatments with increasing NaCl concentrations, [48] noticed a linear decline in the levels of total chlorophyll, chlorophyll a, chlorophyll b, carotenoids, and xanthophylls as well as the intensity of chlorophyll fluorescence in Vigna radiata.Salt stress also leads to change in size and density of stomata, which ultimately results in reduced stomatal conductance. In a study conducted by de Villiers et al., [49] they analyzed photosynthetic and stomatal response to increasing salt stress in Atriplex semibaccata R. Br. The number of chloroplasts in the chlorenchyma and bundle sheath cells decreased as salinity rose and hence also blocks the developing ability of these two cells types. With increasing salinity, the intercellular CO2 concentration rose while the net leaf photosynthetic rate and leaf stomatal conductance decreased. The lower leaf photosynthetic rates were likely due to stomatal closure. According to the stomatal indices, there is a trend towards more stomata per unit leaf area as salinity rises.

2.4 Effect on biomass and crop yield

Salinity reduces crop plant growth and yield by reducing soil moisture availability and due to the toxicity of sodium and chloride ions at high concentrations to the plant. Soil salinity has a negative impact on vital metabolic, biochemical, and physiological processes within plants, resulting in grain quality degradation. The severity of the stress determines the extent of changes in grain quality caused by salinity. Grain quality is affected physiologically due to salt accumulation in the root zone, which induces osmotic stress and vigorously disrupts cell ion homeostasis. Salt exposure causes osmotic stress at first, followed by ion toxicity, which inhibits growth, grain development, and quality, especially if the exposure periods are prolonged. The deterioration of cereal grain quality has also been explained from an agronomic standpoint. The reduction in root water uptake capacity caused by osmotic stress contributes to growth inhibition, decreased crop productivity, and poor grain quality [50]

2.5 Effect on plant growth

One of the initial effects of salt stress is the slowing of plant growth. Salt in soil and water inhibits plant growth for two main reasons. First, it impairs the plant's ability to absorb water, leading to water scarcity during critical developmental stages, which in turn slows growth. This is known as the osmotic or water deficit effect of salinity [51, 52]. Second, salinity causes salt-specific or ion-excess effects, where salts enter the transpiration stream and damage cells in the transpiring leaves, ultimately reducing growth [43, 53]. As explained by the researchers these two effects result in two-phase growth response to salinity. Phase 1: The first phase of the growth response is caused by the effect of salt outside the plant. The salt in the soil solution inhibits leaf growth and, to a lesser extent, root growth. Phase 2: The toxic effect of salt inside the plant causes the second phase of the growth response. The salt taken up by the plant concentrates in old leaves: prolonged transport into transpiring leaves results in extremely high Na+ and Cl.− concentrations, and the leaves die [52, 53]

2.6 Effect on oxidative stress in plants

Electron transport chains (ETC) in chloroplasts and mitochondria can overflow, degrade, or even break down as a result of abiotic and biotic stress in living things, including plants. In these circumstances, molecular oxygen (O2) functions as an electron acceptor, leading to the buildup of reactive oxygen species (ROS). Each of these strongly oxidizing substances—singlet oxygen, hydroxyl radicals (OH), superoxide radicals (O2), and hydrogen peroxide (H2O2) poses a risk to the integrity of cells [54]. As cell membranes are the primary targets of many plant stresses, ROS may disrupt normal metabolism via membrane lipid peroxidation. In plants subjected to salt stress, the process of lipid peroxidation in biological membranes may lead to structural alterations such as the denaturation of proteins and nucleic acids, as noted by [55]. It has been documented that the occurrence of oxidative damage is a consequence of the surpassing of plants' scavenging systems' capabilities by levels of ROS. Elevated ROS levels have the potential to deactivate enzymes and harm crucial cellular constituents, ultimately causing a halt in plant growth and, ultimately, mortality; according to [56]. Numerous botanical investigations have observed an elevation in RO generation in response to saline environments. It has been demonstrated that ROS-induced impairment of membranes plays a significant role in the cellular harm inflicted by salinity on a range of agricultural plants including rice, tomato, citrus, pea, and mustard [43]. Being common components of plant plasma membranes, polyunsaturated fatty acids are particularly exposed to the action of ROS, especially singlet oxygen and hydroxyl radicals. These react with polyunsaturated fatty acids, leading to the formation of lipid peroxides. The changes generated by ROS leads to disturbance in the physicochemical structure of the plasma membrane: it becomes less fluid and its permeability increases, resulting in electrolyte leakage [56].

2.7 Effect on leaf anatomy and ultrastructure

Anatomical modifications induced by salinity could be like change in mesophyll surface area to leaf area, thickening of leaf, increased epidermal thickness or changes in stomatal distribution. Longstreth et al. [57] carried out an early assay in this field,these researchers examined how three plants with various responses to salt stress—Phaseolus vulgaris, which is salt-sensitive; Gossypium hirsutum, which is moderately salt-tolerant and the salt-tolerant Atriplex patula. The halophytic species Atriplex patula exhibited increased leaf thickness due to elevated epidermal and mesophyll thickness following irrigation with different concentrations of NaCl solutions (0.05, 0.1, 0.2, 0.3, and 0.4 M). Subsequently, notable enhancements in succulence measurements were observed. Conversely, the remaining two species, characterized by lower salt tolerance, demonstrated contrasting outcomes. Navarro et al. [58] examined the structural modifications linked to sodium chloride-induced stress in the leaves of Arbutus unedo using optical microscopy on semi-thin slices. A comparison between untreated plants and those subjected to saline conditions revealed that there was no notable change in the size of the palisade cells in the outer layer. Conversely, a marked increase in the size of the palisade cells in the inner layer was observed, corresponding with the varying levels of salinity tested (0 mM, 52 mM, and 105 mM NaCl). These authors also noticed an important decrease in the intercellular spaces in the spongy mesophyll in both saline treatments compared with control leaves, and this decrease affected the stomatal and mesophyll conductance to CO2.

2.8 Effect on nutrient imbalance

It is well known that salinity-induced nutritional disorders can harm crop performance. Nutritional disorders may occur as a result of salinity's effect on nutrient availability, competitive uptake, transport, or distribution within the plant [43]. Numerous studies have found that salinity reduces nutrient uptake and accumulation in plants.

Firstly, salinity affects plant's micronutrient concentrations in distinct ways depending on the crop species and salinity levels. Secondly, plant nitrogen accumulation ability could be severely reduced by salinity. Reduced N2 uptake occurs under saline conditions due to interactions between Na+ and NH4 + and/or Cl− and NO3−, which ultimately reduces crop growth and yield [59]. Moreover, phosphorus availability is also reduced in saline soil. Elevated sodium chloride (NaCl) levels in the root medium reduce nutrient assimilation, particularly of K and Ca, resulting in K, Ca, and Mg ion imbalances [60, 61].

2.9 Effect on antioxidants enzymes

In plants, oxidative stress occurs frequently. Under physiological conditions, all aerobic organisms generate reactive oxygen species (ROS) in their cells, including singlet oxygen (O2), superoxide anions (O2.−), hydrogen peroxide (H2O2), and hydroxyl radicals (HO). The antioxidant system regulates the levels of these ROS [62, 63]. Stressful environments cause a significant increase in their generation, which modifies antioxidant potential. It has been evident in recent years that ROS plays a dual role in plants, acting as both toxic substances and important regulators of a range of biological processes, including development, cell cycle, growth, programmed cell death, hormone signaling, and abiotic cell responses. In one of the studies, researchers found that salinity stress-induced alterations in the cellular antioxidant pool, elevating levels of total carotenoids, ascorbate, proline, total polyphenol content (TPC), total flavonoid content (TFC), and total antioxidant capacity (TAC) in both VA14 and VA3 varieties of A. tricolor [64].Moreover, high salt concentrations can also result in ionic stress, where excessive sodium (Na+) and chloride (Cl−) ions accumulate in plant tissues, disrupting cellular functions and nutrient balance. Such ionic imbalances can be critical, highlighting the need for developing salt-tolerant crop varieties to sustain agriculture in saline-prone areas. These conditions impair photosynthesis, reduce leaf area, and stunt overall plant development. This impairment not only affects individual plants but also threatens food security on a larger scale, emphasizing the importance of managing soil salinity.

3 CRISPR tool kit

Despite CRISPR’s discovery in 1987 [65], the role of CRISPRs in bacteria wasn't understood until 2005 [66, 67], and it wasn't until 2007 that Cas-independent CRISPRs were found to confer immunity [68]. Before the emergence of CRISPR, various gene editing instruments like meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) were employed. CRISPR represents the most recent advancement in gene-editing (GE) technology, with broader applications in plant research [69]. CRISPR and Cas proteins are components of natural adaptive defense systems against invading viruses in archaea and bacteria. CRISPR/Cas loci in bacteria are made up of a CRISPR array, which can include hundreds of direct, frequently palindromic repetitions (35–45 bases) separated by unique spacer sequences (30–40 bases) [70]. In the process of GE technology, the two DNA repair mechanisms namely, homology-directed repair (HDR) or non-homologous end joining (NHEJ) have been utilized by the cell to repair the DSBs. The HDR mechanism makes use of long homologous DNA sequences for the precise insertion at the target site and hence results in more specificity, while the latter NHEJ simply utilizes the broken ends and performs the error-prone repair that ultimately results in non-specific mutations. Although being non-specific, the NHEJ repair system is more common [71, 72].

The CRISPR/Cas system generates an immune response in three stages: adaptation, pre-CRISPR RNA (crRNA) expression/processing, and interference. A short synthetic guide RNA (gRNA) sequence directs Cas9 to the specific genomic target site, which is further determined by the presence of a protospacer-adjacent motif (PAM) sequence immediately following the target site [73]. CRISPR/Cas is being used for gene editing in a variety of crop species, introducing agricultural traits of great value into many of them [74]. Because of their ability to induce precise nucleotide changes, the newly developed precise CRISPR/Cas technologies, in particular, promise to have a significant impact on agriculture. CRISPR/Cas, on the other hand, is capable of much more than just editing specific loci for crop improvement. With the advance technological interventions, there has been promising evolution in this technology such as prime editing, base editing, PAMless editing, Drosha based editing that possess multiple targeting efficiency [75]. These recent interventions have proven to be highly proficient, cost-efficient as well as user-friendly. Based on this versatile game-changing platform, a number of novel plant biotechnologies capable of promoting gene regulation and protein engineering have emerged. This editing holds promise for a major revolution in basic and applied plant biology research in the ever-evolving climatic conditions [76]. These technologies have already had an impact on basic biological research and have raised the possibility of widespread application. Using CRISPR/Cas9 genome editing, it is possible to improve crops using targeted gene editing to enhance specific traits. It can be used to knock out genes that negatively affect a plant’s ability to tolerate high salinity. By deactivating these genes, plants can be made more resilient. For example: SOS Pathway: The Salt Overly Sensitive (SOS) pathway in plants involves several genes like SOS1, SOS2, and SOS3. Knocking out negative regulators of this pathway can enhance salt tolerance [32, 77,78,79]. It has been used to target genes involved in sugar metabolism, hormone transport, posttranslational modification, transcriptional regulation, protein stability, and signal transduction, according to reports published in the last two years. However, given the polygenic nature of the salt tolerance trait, this progress is insufficient, and more molecular entities must be targeted. There are numerous tools available for editing not only DNA but also RNA [80]. These tools are classified into three categories: knockout, base editing, and allele exchange/replacement. The most common method is targeted mutation, though it is frequently impossible to predict which mutation will occur. A stop codon is frequently generated in the nucleotide sequence, resulting in the target product's non-functionality. The following step entails targeted base exchange or targeted integration of foreign DNA (knock-in). The key SNP from the functional allele can be introduced into the homologous allele of salt-sensitive elite plant varieties using precise base-editing. In one of the study, [81] demonstrated that for the survival of salt-tolerant Pokkali and wild rice halophyte, Oryza coarctata, amino acid substitutions (D332H, V395L) impacted High-affinity Potassium Transporters (HKT1;5) transporters that are more effective than those of salt-sensitive IR29 in extracting Na+ from the vascular system.

Recently, researchers have discussed the key nutrient transporters involved in the acquisition and redistribution of nutrients from the soil. They provide insights into the potential application of the CRISPR/Cas system to improve nutrient transport in plants that could be achieved by engineering key residues of nutrient transporters, modifying the transcriptional regulation of nutrient transport signals, and engineering motifs in promoters and transcription factors [82]. Technology for gene editing such as CRISPR/Cas9 show promise for improving crop nutrient use efficiency (NtUE) and nutrient stress tolerance. Gene editing, by targeting negative regulators of nutrient signaling, has the potential to improve nutrient uptake and stress response under resource-limited conditions. These technologies have been used to develop genome-edited crops with improved nutritional properties, such as high-GABA (Gamma-aminobutyric acid) tomatoes and high-oleic acid soybeans. These edited crops are nearly identical to naturally occurring mutations, which aids in their market acceptance [83]. Antony Ceasar et al., [84] compiles a comprehensive list of the functional residues of various macro- and micronutrient transporters involved in nutrient acquisition and redistribution in plants. They have also used bioinformatics tools to analyses these functionally important residues. Based on their findings, they provide insights into the potential use of CRISPR/Cas tools to edit key residues in order to improve nutrient transport and agronomical performance.

Furthermore, Specific genes that contribute to salt stress tolerance can be modified to enhance their functionality. For example, NHX1: Altering the NHX1 gene, responsible for encoding a sodium/hydrogen exchanger, has the potential to enhance the plant's capacity to sequester sodium ions into vacuoles, consequently diminishing their harmful impact in the cytoplasm [85, 86]. The utilization of CRISPR can streamline the incorporation of novel genes from salt-tolerant species into agricultural crops. This transfer of genes horizontally can equip crops with novel traits; for instance, genes from salicornia plants, which thrive naturally in high-salinity environments, can be transferred to conventional crops to enhance their salt tolerance [87]. Various CRISPR-Cas9 toolkits for genome editing are well explained in the Fig. 3.

Fig. 3
figure 3

CRISPR-Cas9 toolkit for genome editing. ABE, adenine base editors; Cas, CRISPR-associated; CBE, cytosine base editors; CRISPR, clustered regularly interspaced short palindromic repeats; CRISPRi, CRISPR interference; CRSPRa, CRISPR activation, PAM; protospacer adjacent motif

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3.1 Base editing

It has been established that CRISPR/Cas9 is widely used for various purposes; however, their applications for precise and sophisticated gene editing are limited. The process of targeting precise point mutations or single-base conversions (base editing) remains difficult. Using base editing or a specific base conversion, a target base pair can be replaced with another base pair without causing DNA damage [53, 88]. There exist two base editing systems namely, adenine base editors and cytosine base editors. Further, base editing is more specific than CRISPR/Cas9 because the off-target mutation rate is lower specifically in adenine base editing in comparison to cytosine base editing [89,90,91]. However, the low editing efficiency of HDR has limited its use in plants [92, 93]. Numerous genes in plant species have undergone alterations through various Biological Entities (BEs). Consequently, this has resulted in the precise induction of mutations, including stop codons, modifications in amino acids, and alterations in regulatory sites, within the genomes of rice, wheat, maize, potato, watermelon, cotton, tomato, and Arabidopsis [94, 95].

3.1.1 CBEs (cytosine base editors)

CBE comprises a Cas9 nickase (nCas9) containing the D10A mutation, leading to the inactivation of RuvC (a Cas9 nuclease domain), coupled with two proteins: a cytidine deaminase and an inhibitor of uracil DNA glycosylase (UDG), namely UGI. C:G > T substitution is induced by CBE, where a nucleotide transition occurs at specific DNA loci recognized by a singular guide RNA.. The deaminase enzyme facilitates the conversion of cytidines to uridines within the non-target strand, which constitutes the single-stranded DNA (ssDNA) segment of the R-loop generated by the nCas9 (D10A)-sgRNA complex. Meanwhile, the UGI protein acts to inhibit the conversion of cytidines to apurinic/apyrimidinic (AP) sites by the uracil DNA glycosylase (UDG). Upon introduction of nCas9 (D10A), a nick is generated on the target strand, prompting the activation of the DNA mismatch repair pathway, or potentially other DNA repair pathways. This activation leads to the preferential resolution of the U: G mismatch into the desired U:A, eventually resulting in a T:A product following DNA replication, consequently causing a C:G > T:A base transition. The efficiency of this base editing technology has led to the optimization and extensive development of CBE systems across a diverse range of plant species. According to the study, base editing windows of positions 1 through 17 in the protospacer can be opened by CDEs based on Perkinsus marinus CDA1 or human AID and CBEs based on human APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic subunit 3A), as they both exhibit high base editing efficiency without motif preference) [94, 96, 97]. Also, recently rice plants were examined using two newly developed CBEs based on rationally designed truncated human APOBEC3B (hAPOBEC3B) with high specificity and precision [98]. By using the cytosine base editor to introduce a point mutation into the CENH3 gene (Centromere Specific Histone 3), the haploid induction rice line was created (CBE) [99].

3.1.2 ABEs (adenine base editors)

ABEs use adenosine deaminase as an effector, fused with nCas9 (D10A), to expand base editing to include A: T > G:C substitutions [100]. ABEs were later developed to convert A to G. ABEs, unlike CBEs, do not require a DNA glycosylase inhibitor, and natural adenine deaminase cannot accept DNA. Liu's team created several generations of ABEs. According to Li et al., [101] the ABE system can be used to efficiently introduce point mutations in plants. Six rice genes Acetyl-coenzyme A carboxylase (ACC), ALS, CDC48, DEP1, NRT1.1B, and OsEV) were successfully edited using an ABE. Hua et al., [102] investigated the efficacy of various ABEs using the IPA1 (OsSPL14), OsSPL17, OsSPL18, and SLR1 genes. Two ABEs, ABE7.8 and ABE7.10, were used to edit the genes MPK6, MPK13, SERK2, WRKY45, and Tms9-1 [103, 104]indicating that base editing tools can be used to improve crop yield. Based on the editing of the OsACC, OsALS, OsDEP1, OsNRT1, OsCDC48, and OsWx genes in rice, ABEs had higher mutation efficiency than CBEs [105] Numerous different studies [90, 106, 107] have reached similar conclusions. Furthermore, some studies of ABE mutation efficiencies found that unwanted mutations were less common with ABEs than when CRISPR/Cas9 tools were used [108] Another study used ABE7.10 to expand base editing in rice and wheat by targeting DEP1, TaEPSPS, and GW2 genes [102].The same strategy was used to edit four Arabidopsis genes (AtALS, AtPDS, AtFT, and AtLFY) and two rapeseed genes (BnALS and BnPDS) [109].

3.2 Transcription R (regulation)

Plants can undergo stress tolerance changes when they are exposed to adverse conditions such as salinity, as a result of upregulation or downregulation of various genes with various functions [110]. It has been found that many of these genes upregulated or downregulated encode proteins that are crucial to stress-related growth or metabolic changes [111, 112], while others encode regulatory proteins like TFs (Transcription factors), which regulate salt-sensing and signal transduction pathways as well as the expression of salinity stress-related genes. Researchers have developed transgenic plants with modified TF expression to improve salt tolerance [113]. Numerous studies on various plant species have demonstrated that salt stress responsive genes are controlled by stress-responsive transcription factors together with their promoter regions [114,115,116,117,118,119,120]. The molecular mechanisms and associated genes that are involved in plant salinity tolerance is well documented in several reviews [26, 27, 121]. Another method of controlling gene expression without introducing mutations is made possible by CRISPR activation/interference (CRISPRa/CRISPRi) tools. The CRISPR-fused-cargo has been delivered to the targeted genomic region by fusing a variety of effector molecules (either gene activators/gene repressors) to partly impaired (nCas9) or nuclease-deficient Cas9 (dCas9).

3.2.1 CRISPRa (CRISPR activation)

The CRISPR/dCas9 system can also be used for gene activation (CRISPRa), depending on the effector attached to the dCas9.It has been shown that dCas9-VP64, directed by sgRNA, can recruit transcription factors and regulate gene transcription when it binds to a target gene's promoter. As a necessary consequence; it has the potential to replace traditional GM-based gene silencing and overexpression methods. Because gene editing tools allow for precise modification or regulation of a gene of interest, they have an advantage over genetic engineering methods in which transgene integration is random (in the case of overexpression), which can repress or activate other genes, and unwanted silencing of other genes can occur via siRNA-mediated RNAi. To use dCas9 activators or repressors to activate or repress a target gene, sgRNAs should be directed to the promoter region of the gene of interest (GOI) [122]. CRISPR-dCas9-based transcriptional regulation experiments in plants typically require the expression of individual gRNAs and dCas9-effector fusion proteins from a single T-DNA [123, 124]. CRISPRa was used to activate the gene Arabidopsis vacuolar H+-pyrophosphatase (AVP1). In CRISPRa plants, the expression of the AVP1 gene was two to five times higher. AVP1 activated plants displayed more leaves, more leaf area, and phenotypes like those of AVP1 overexpression transgenics [125]. Li et al., [126] showed that dCas9 transcriptional activation system can be multiplexed in cell-based assays when co-expressed with three sgRNAs targeting WRKY30, RLP23, and CDG1. All three genes showed significant increases in endogenous gene expression as measured by RT-qPCR. Scientists successfully tested the efficiency of the CRISPR-Act2.0 system in simultaneously activating three independent endogenous genes in rice protoplasts, Os11g35410, Os03g01240, and Os04g39780. The analysis of rice protoplast transformants revealed that CRISPR-Act2.0 is a more effective system for gene activation than the dCas9-VP64 system. The successful demonstration of real-time gene activation with CRISPR-Act2.0 suggests promising applications for future plant research.

3.2.2 CRISPRi (CRISPR interference)

In CRISPRi, dCas9 binds to a gene's target site and prevents transcription from starting. It is true that dCas9 by itself can interfere with the transcriptional machinery, but it is also true that its effectiveness depends on the repressors that are involved. Researchers have found that dCas12a (also known as dCpf1) is more effective at repressing transcription than dCas9 Comparatively to dCas9, dCas12a can process a single transcript tandem crRNA array into multiple crRNAs on its own. CRISPRi has been used to achieve effective, long-term RNA-guided transcriptional suppression of a target gene. Transcription suppression is inducible and reversible, and target recognition is solely dependent on the sgRNA sequence. Recruitment of dCas9 to the sgRNA recognition complex inhibits gene expression by interfering with transcriptional elongation, RNA polymerase binding, and transcription factor [127,128,129]. The transcription repression domain of transcription effector proteins is used by the interference mechanism. When SRDX effector (a repressor protein) and dCas9 were combined for repression, the transcription activity of PDS was considerably decreased in comparison to the control (dCas9 alone) [130].

3.3 Multiplexing

Another CRISPR characteristic called multiplexing makes it possible to modify polygenic traits—or numerous traits—at once [131]. Multiplex editing is one of CRISPR's main advantages, enabling the editing of multiple targets at once [132]. It can be accomplished in two ways: by using a single promoter to express multiple sgRNAs simultaneously, or by using individual promoters for each sgRNA [133]. In order to improve crop productivity, quality, and stress tolerance, CRISPR/Cas multiplexing has been successfully used to date. As a consequence of several tests, it has been determined that new methods and applications are steadily emerging. It involves the expression of multiple guide RNAs or Cas enzymes simultaneously for gene editing and transcriptional regulation.

One such multiplexing system is: tRNA-mediated multiplexing. The idea behind tRNA-mediated multiplexing is that the endogenous RNA-processing apparatus is responsible for producing and digesting this essential cellular component, several tRNAs–gRNA are created based on this idea [134]. These tRNAs can then be processed by RNaseZ and RNaseP, which cleaves the tRNAs' 5′ and 3′ ends and releases the individual gRNAs with the necessary 5′ targeting sequences. The Cas9 protein is thus properly directed by these desired gRNAs to edit various chromosomal sites. Since RNase P and Z are present in every domain of life, a wide variety of gRNAs can be processed from tRNA–gRNA arrays in a wide range of organisms. A single Pol II promoter in rice protoplasts might produce numerous gRNAs and an endonuclease (encoded in an exon) by using tRNA–gRNA arrays, which can also be encoded in eukaryotic introns and processed by the spliceosome complex [135]. This multiplexing method works well on a variety of organisms and is very efficient, specific, stable, and uniform. The Pol III promoters (such as U3p) regulate the transcription of poly-tRNA-gRNA, but unlike other gRNAs, these PTGs do not require a particular nucleotide at the outset of transcription. Consequently, it is possible to efficiently use every CRISPR/Cas9 sgRNA expression vector to express PTGs at several targets. Multiple mutations can be simultaneously induced at different genomic loci thanks to the PTG technology.

Furthermore, the Pseudomonas aeruginosa Cys4 system is a potent tool for multiplexed genome editing and has demonstrated the capacity to quickly and efficiently regulate 20 genes in Saccharomyces cerevisiae. gRNA arrays can be produced from Pol II or Pol III promoters and removed by endonucleases of the Cas family, like Csy4, which in some native CRISPR systems processes pre-crRNAs [136]. RNA transcripts contain a 28-nt stem-loop region that Csy4 recognizes and cleaves after nucleotide 2033. Multiple gRNAs can be generated and processed from a single promoter in mammalian cells, yeast, and bacteria by surrounding each gRNA in an array with the Csy4 recognition sequence [137,138,139]. Even while Csy4-mediated gRNA array processing makes it possible to process many gRNAs from a single RNA transcript, co-expression of Csy4 might occasionally be unfavorable due to cytotoxicity at high concentrations [140].

3.4 RNA editing

The recent discovery of RNA-targeting Cas13 (formerly C2c2) effectors and the DNA-targeting Cas12 (formerly Cpf1) effector indicates there are additional potential tools in addition to CRISPR/Cas9 [141,142,143]. The Type VI effectors are those that target and cleave RNA, with Cas13a being the most well-studied [142, 144, 145]. The effective use of Cas13 in RNA investigations, including viral interference, RNA knockdown, and RNA detection in different species, has been described in a number of publications. As most plant viruses are RNA viruses, Cas13 was also utilized to breed viral resistance in plants [146]. The use of CRISPR/Cas13 in investigations of plant RNA biology, however, is still in its early stages (Kavuri et al., 2022).When Cas13a from Leptotrichia buccalis (LshCas13a) was successful in conferring resistance to one of the RNA viruses, Turnip Mosaic Virus (TuMV) in Nicotiana benthamiana [147]. Apart from this, scientists endorsed the utility of this tool in virus detection [148]

3.5 Prime editing

Prime editing is a sophisticated and adaptable method of genome editing that was created to alleviate some of the shortcomings of previous CRISPR-Cas9 procedures. Unlike regular CRISPR-Cas9, which uses double-strand breaks in DNA to generate edits, prime editing provides a more accurate and potentially safer approach of producing targeted genetic modifications [149, 150]. Prime Editing's key components include a Prime Editor Complex and a Prime Editing Guide RNA (pegRNA). Prime editing employs a fusion protein that combines a modified Cas9 endonuclease (also known as "nicking" Cas9 or Cas9 nickase) and a reverse transcriptase enzyme. Cas9 nickase causes a single-strand break (nick) in the DNA rather than a double-strand break. Prime Editing guide RNA is a customized RNA that not only directs Cas9 to the target location but also contains a template for the desired genetic alteration. The pegRNA consists of two parts: i. a guide sequence that guides the Cas9 nickase to a specific DNA region, and ii. an extension that includes the sequence to be incorporated into the genome, flanked by homology arms that match the target DNA site [151].

Prime editing encompasses a variety of approaches and techniques, which are mainly identified by the particular arrangements of the constituent parts and the type of modifications they aim to implement. These are the primary categories and features of prime editing:

3.5.1 Types of prime editing

  1. 1.

    PE1 (Prime Editor 1) combines a reverse transcriptase (RT) with the Cas9 nickase (H840A). It introduces single-strand nicks into the DNA and extends the strand of the DNA with the edit using the RT.

  2. 2.

    PE2 (Prime Editor 2): Using an enhanced reverse transcriptase, it is comparable to PE1. It improves the effectiveness of adding the intended modifications to the DNA.

  3. 3.

    PE3 (Prime Editor 3): It builds on PE2 by introducing a single guide RNA (sgRNA) to create a second nick on the non-edited strand. This twin nicking method improves the installation of the intended edit by activating DNA repair mechanisms that promote the edit's integration [152].

By focusing on important genes implicated in salt stress responses, such as those that encode proline-rich proteins, primary editing can bring positive changes that improve the plant's ability to survive salinity. The use of prime editing in crops like rice and wheat has shown encouraging results in generating salt-tolerant cultivars, hence enhancing agricultural resilience and production in the face of abiotic challenges such as salinity [153,154,155].

3.6 CRISPR-directed evolution

CRISPR-directed evolution is a novel application of the CRISPR-Cas system that takes advantage of its precision and versatility to accelerate the process of directed evolution. Directed evolution replicates the natural selection process by evolving proteins or nucleic acids toward a user-defined goal at a faster and more specific rate thanks to human involvement [156]. When paired with CRISPR, directed evolution becomes an effective method for genome editing, allowing organisms to generate novel characteristics and functionalities. CRISPR-Directed Evolution allows researchers to uncover various single guide RNA (sgRNA) variations that bind successfully to Cas9, improving DNA cleavage and editing efficiencies at specific target sites. This method enables the generation of tailored sgRNA sequences that can target and change genes involved in stress regulation networks, ultimately leading to the establishment of plants that are highly resistant to salt stress and other environmental problems [157].

3.6.1 Techniques and methodologies

  1. 1.

    CRISPR Mutagenesis: CRISPR can introduce targeted mutations at specific genomic sites, resulting in a wide set of variations. These libraries are then tested for desirable characteristics, which aids in the directed evolution process.

  2. 2.

    Adaptive Laboratory Evolution (ALE): Using selection pressure in a controlled setting, organisms can develop to acquire advantageous features. CRISPR can increase genomic variation, whilst selective pressure ensures the survival and dissemination of beneficial mutations [158, 159].

4 CRISPR/ Cas targets for salt stress tolerance

One of the most significant abiotic stresses affecting global crop production is salinity. More than 50% of agricultural fields might become dangerously salinized by the year 2050. The most cost-effective and environmentally friendly approach to salinity control is the cultivation of salinity-tolerant cultivars. Breeding stress-tolerant plants can improve crop yield under stress conditions, whereas CRISPR/Cas9 gene editing has been shown to be an efficient method for molecular breeding to improve agronomic traits in crops, including stress tolerance [160]. The significant advancement of the CRISPR/Cas gene editing tool, combined with the incorporation of advanced approaches, has enabled biologists to edit genomes with greater efficiency and precision never before imagined. The CRISPR/Cas editing module has opened up new avenues for editing plant genomes to improve desired traits and develop varieties resistant to various stresses. The CRISPR/Cas technique for trait improvement has been used successfully to edit crop plant genomes such as maize, rice, tomato, soybean, and sorghum [161,162,163,164,165,166,167,168]. The gene targets for CRISPR/ Cas system for salt tolerance in various plants are elaborated in the (Table 1).

Table 1 CRISPR gene targets to enhance salt stress tolerance in plants
Full size table

Plants use a wide range of mechanisms to upregulate or downregulate (increase or decrease) the production of specific gene products in response to salinity stress (protein or RNA). At Central Dogma, various mechanisms of gene regulation were discovered, ranging from transcriptional initiation to RNA processing and protein posttranslational modification. Transcriptomic analysis provides detailed information about gene expression at the mRNA level, which is commonly used to screen for candidate genes involved in stress responses (Fig. 4). Encoding, cloning, and characterizing important genes rely heavily on genomic approaches. Using transcriptomic and genomic approaches, a large number of salt-responsive transcription factors and genes that are either upregulated or downregulated in response to salinity stress have been identified and characterized [111]. Only a handful of genes involved in CD have been so far targeted and characterized using CRISPR to understand the molecular mechanisms regulating plant salt tolerance, which can be broadly classified into following categories [13, 206]

Fig. 4
figure 4

Scheme illustrating plant responses the salt stress using CRISPR/Cas targets

Full size image

4.1 Transcription factors

Numerous genes have been found and described by CRISPR/Cas-based gene editing to increase plant salt tolerance [150, 153, 162]. In numerous crops, the use of CRISPR methods for promoter engineering has been studied. Improving the control of a targeted gene's expression under salt stress may be possible by simultaneously altering a promoter and its associated TFs. The OsRAV2's salt response is directly regulated by the GT-1 element. This research advances knowledge of the potential roles of OsRAVs and the molecular processes that control plant gene expression in salt stress [188]. In addition, it would be intriguing to see how changed promoter design might produce crops that are resistant to salt [207].

During salt stressors, the two TF AtWRKY3 and AtWRKY4 genes mutants showed a considerable up-regulation of stress-related genes such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) in Arabidopsis [198]. Salt stress resistance was seen in rice when SnRK2, osmotic stress/ABA-activated protein kinases (SAPK-1, SAPK-2) were mutated by CRISPR/Cas [208]. Knockout of Acquired Osmo-tolerance (AtACQOS) gene provided tolerance against salt stress in Arabidopsis [197]. In two separate investigations, salt tolerance in soybean was imparted by knockout mutants of the genes for abscisic acid (ABA)-induced transcription repressors (AITRs) and GmDrb2a, GmDrb2b. These mutant plants produced longer roots and shoots than WT plants and displayed increased ABA sensitivity [169, 170]. CRISPR/Cas9 gene editing was used to create transgene-free Gmaitr mutants. In response to salt treatment, Gmaitr mutant seeds germinated greater than wild type seeds, and mutant plants in the normal soil field exhibited morphological similarities to wild type plants but flourished in the salty soil field. The AITR gene may be one of the targets for CRISPR/Cas9-mediated mutagenesis to improve salt stress tolerance in a variety of plant species. CRISPR mutants with loss-of-function of SnRK2 and osmotic stress/ABA-activated protein kinases SAPK-1 and SAPK-2 genes showed resistance to salt stress in rice [186]

The OsRR22 gene, encodes a 696-amino acid B-type response regulator transcription factor involved in both cytokinin signal transduction and metabolism. CRISPR/Cas mediated knockout lines has been shown to increase salt tolerance significantly [209]. A precise editing of a 20-bp nucleotide sequence in the first exon of OsRR22 gene in rice variety WPB106 exhibited improved growth at 0.75% NaCl concentration [180]. In rice, 158 NAC transcription factors have been identified as being involved in a variety of abiotic stresses. The CRISPR/Cas9 system was used to perform targeted mutagenesis of the OsNAC041 locus in order to determine the specific role in salinity stress. When treated with 150 mM NaCl for 7 days, seed germination and subsequent growth of the Osnac041 mutants were suppressed compared to the wild type. Moreover, under salt stress, the shoots of the Osnac41 mutant seedlings were shorter than those of the wild-type seedlings, indicating that the mutants were sensitive to salt stress [181]. The ability to withstand salt stress is improved by CRISPR/Cas9-mediated knockout of the OsbHLH024 transcription factor in rice [173]. OsRST1 encodes the AUXIN RESPONSE FACTOR 18 protein (OsARF18), which inhibits the transcription of the asparagine synthetase 1 (OsAS1) gene to adversely control nitrogen metabolism. By encouraging asparagine formation and preventing excessive ammonium (NH4+) accumulation brought on by salt stress, genome-editing of OsRST1 boosted the expression of OsAS1 and improved nitrogen (N) usage, enhancing the ability to live in saline environments [189]. The salinity-induced OsRAV2 promoter contains the GT-1 element (GAAAAA), which is important. The OsRAV2 promoter's GT-1 element was directly contacted by the trihelix transcription factor OsGTγ-2. OsGTγ-2 was mostly expressed in roots, sheaths, stems, and seeds, had a nucleus-specific target and was activated by salinity stress. OsGTγ-2 controlled the transcription of several important ion transporting genes, including OsHKT2; 1, OsHKT1; 3, and OsNHX1 by interacting with their promoters. Together, these findings indicate that OsGTγ-2 is a key positive regulator of rice to salt stress and may play a role in controlling rice's ability to adapt to salinity [177]. A different member of the TFs family called BEAR1 controls rice's ability to tolerate salinity. Under salt stress, rice that had BEAR1 mutated using CRISPR/Cas9 showed notable alterations. BEAR1 is mostly expressed in roots, seedling stages, and spikelet’s and is triggered by salt stress. By controlling the expression of salt-responsive genes and ions transport, BEARI improved rice's resistance to salinity [190].

There have been a number of genome-editing studies in rice for salt stress. To confer rice salinity resistance, CRISPR/Cas has been used to knock off a number of transcription factors, including OsDST [176], OsNAC45 [74], OsNAC3 [163], AGO2 [179], OsDOF15 [38], OsSLR 1, OsPIL14 [178], OsBGE3 [210], OsVDE ( [172], OsFLN2 [89], OsMADS27 [191], OsSPL10 [183], OsPP65 [192] and OsBBS1 [187]. By changing at least one allele of the coilin gene, it was possible to significantly improve the edited lines’ ability to withstand salt stress in potatoes [210]. High salinity tolerance was achieved in tomato at the germination and vegetative stages by precisely removing the SlHyPRP1 negative-response domain(s) [193]. Also, by editing tomato SlARF4 and barley HvITPK1, HvHVP10 utilizing CRISPR/Cas9 technology, crops' capacity to withstand salt stress is significantly increased [194, 203, 204, 211]. The Alkali Tolerance 1 (AT1) locus encodes an unusual G protein γ subunit that influences the efflux of hydrogen peroxide (H2O2) under stress, hence influencing alkaline sensitivity. It is possible that AT1 has a detrimental effect on plant alkali tolerance because its overexpression lowered alkaline tolerance in sorghum and rice and caused a more pronounced alkaline sensitive response. Nevertheless, deletion of AT1 boosted alkaline stress tolerance in sorghum, millet, rice, and maize, suggesting a conserved mechanism in monocot crops [205]. A protein called ZmESBL, encoded by ZmSTL1, is localized to the Casparian strip (CS) domain. Mutants lacking ZmESBL exhibit a faulty CS barrier due to poor lignin deposition at the endodermal CS domain and become salt hypersensitive [160]. Under salt stress, SlABIG1 deletion greatly increased tomato salt tolerance, increasing root dry weight, proline, and chlorophyll content while lowering ROS, MDA, and Na+ accumulation. Together, these findings showed that SlABIG1 is a critical factor in tomatoes' ability to withstand salinity stress and offered a candidate novel gene for future salt-tolerant tomato varieties [195] (Table 1).

4.2 Epigenetic regulators

Epigenetics is the study of heritable and stable changes in gene expression resulting from alterations in the chromosome rather than changes in the DNA sequence. Although these modifications do not change the DNA sequence itself, epigenetic mechanisms can regulate gene expression through chemical modifications of DNA bases and changes to the chromosomal structure in which DNA is packaged [212,213,214]. Plants are sessile organisms that must adjust to ever-changing environmental conditions. Unpredictable climate changes expose plants to a variety of abiotic stresses. Investigating the regulation of stress-responsive genes can reveal how plants adapt to these changing conditions. Dynamic changes in epigenetic marks, such as histone modifications and DNA methylation, are known to control gene expression in response to environmental stimuli [213, 215, 216]. The effects of epigenetic regulation on gene expression depend on both the types of epigenetic marks and their specific locations on the genes. For example, heterochromatic marks such as DNA methylation and H3K9me2 can repress gene expression by inhibiting transcription when present in the promoter region [217]. Moreover, DNA methylation at the 5' position of cytosines (5mC) is a significant epigenetic marker. Plants methylate DNA in all cytosine sequence contexts, including CG, CHG, and CHH (where H stands for A, T, or C) [202]. High salinity, which causes ion toxicity (primarily Na+), hyperosmotic stress, and secondary stresses such as oxidative damage, inhibits plant growth and development and poses a significant challenge to plant agriculture worldwide [1]. Also, high Na+, low K+, excess Mg2+, and high pH trigger cytosolic Ca2+ signals, which activate the following pathways: SALT OVERLY SENSITIVE3-(SOS3-SOS2), Ca2+−CALCINEURIN B-LIKE PROTEIN –(CBL1/9)- CBL INTERACTING PROTEIN KINASE (CIPK-CIPK23), CBL2/3-CIPK3/9/23/26, and SCaBP1-CIPK11/14. These pathways phosphorylate and regulate the activities of SOS1 (Na+/H+ antiporter), Arabidopsis K+ TRANSPORTER (AKT1, K+ channel), a putative Mg2+ transporter, and H + ATPase, respectively. In Arabidopsis, HKT1 mutation could suppress the salt hypersensitive phenotype of sos3 plants [218]. For instance, bread wheat (Triticum aestivum L., BBAADD) is a typical allohexaploid species that is salt tolerant in comparison to its tetraploid wheat progenitor (BBAA). TaHAG1 gene mutations created by CRISPR-Cas9 in wheat plants showed improved salt tolerance [201]. TaHAG1, a histone acetyltransferase, plays an important role in hexaploid wheat salt tolerance. TaHAG1 overexpression, silencing, and CRISPR-mediated knockout confirmed its role in salinity tolerance. TaHAG1 aided salt tolerance by modulating the production of ROS and signal specificity. Furthermore, TaHAG1 directly targeted a subset of genes involved in hydrogen peroxide production, and TaHAG1 enrichment resulted in increased H3 acetylation and transcriptional upregulation of these loci under salt stress. Tahag1 mutants were created using the CRISPR/Cas9 system to further validate TaHAG1's role in salt tolerance. Under salt stress, the wild-type and mutant plants showed clear physiological differences, such as a decrease in root length and fresh weight, an increase in chlorotic leaves, and a higher Na+ content in the mutants. This suggests that TaHAG1 plays an important role in enhancing hexaploid wheat salt tolerance [201].

4.3 Post-transcriptional regulators

Post-transcriptional regulators, such as microRNAs (miRNAs) and RNA-binding proteins (RBPs), play critical roles in plant stress tolerance by controlling mRNA stability, translation, and localization. These regulators can fine-tune gene expression in response to stress, allowing for rapid and dynamic adjustments in protein synthesis. For example, specific miRNAs may degrade mRNAs that encode stress-related proteins, ensuring that stress response proteins remain at optimal levels. This precise regulation enables plants to adapt and survive a variety of stress conditions, including salt stress. The biological functions of circular RNAs (circRNAs), a significant family of non-coding RNAs, are mainly unknown in plants. OsmiR535 and four circle RNA loci (circRNA) were knocked out using the CRISPR/Cas9 system, increasing rice's tolerance to salinity. Four circRNA loci (Os02circ25329, Os06circ02797, Os03circ00204, and Os05circ02465) were subjected to CRISPR/Cas9-mediated mutagenesis, which demonstrated the involvement of circRNA in the salt response during seedling stage [219]. The circRNA locus Os05circ02465 null mutant in particular displayed great salt tolerance. The circRNA locus Os06circ02797 acts to bind and sequester OsMIR408, a significant and conserved microRNA in plants, according to additional molecular and computational investigations. For the first time in plants, genetic proof of the function of circRNAs in abiotic stress response is presented in this study. The OsMIR528 gene, which codes for a miRNA, was recently discovered to play a role in rice's ability to tolerate salt stress. Although the OsMIR528 gene was altered at the DNA level, it would be intriguing to apply RNA editing techniques in further research [188]. MIR408b-overexpressing transgenic maize is particularly vulnerable to salt stress. ZmLAC9 is a miR408 target. MIR408b knocked mutants were resulted in greater lignin buildup, thicker pavement cell walls, and enhanced maize salt tolerance. Taken together, these findings suggest that miR408 negatively regulates salt tolerance and controls secondary cell wall formation which adversely influences salt tolerance [220].

4.4 Post-translational modifications of proteins

Most protein molecules undergo PTMs throughout the course of their existence. PTMs, which include phosphorylation, acetylation, myristoylation, carbonylation, ubiquitination, sumoylation, and glycosylation, control several aspects of a protein's destiny, including its localization, stability, function, interaction with other proteins, and destruction. Many biological functions, including photosynthesis, disease resistance, and stress tolerance including salinity are modulated by PTMs. CRISPR-mediated PTM alterations have received relatively little attention to yet. Recently, the functions of certain SUMOylation components in rice under salt stress were elucidated by CRISPR/Cas9 targeting of a SUMO protease and Overly Tolerant to Salt 1 (OsOTS1). CRISPR/Cas9 caused effective gene deletion, with 95% of transgenic plants exhibiting the expected outcomes with no off-target consequences [221]. NCA1 (NO CATALASE ACTIVITY 1) is a chaperone protein that regulates catalase (CAT) activity by maintaining the folding of CAT. This protein is encoded by two separate genes in rice, NCA1a and NCA1b. CRISPR/Cas9 was used to create single and double mutants of rice. Under both normal and salinity stress conditions, the single mutants showed no difference in phenotypes and CAT activities when compared to WT, whereas the double mutants consistently displayed very low CAT activity (about 5%) and severe damage when compared to WT [184].

4.5 Protein degradation

Ubiquitin is important in modulating plant responses to a variety of abiotic stresses [222]. An enzymatic conjugation cascade precedes Ub, which includes three types of enzymes: ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Because of its ability to recognize target proteins, E3 ligase facilitates the transfer of ubiquitin to the substrate protein. A multi-ubiquitin chain is conjugated to a substrate protein, and the targets may direct the substrate to the 26S proteasome for proteolysis [223]. Knocking out the Rice PARAQUAT TOLERANCE 3 (OsPQT3) gene, which encodes an E3 ubiquitin ligase, using CRISPR-Cas9 technology significantly improves resistance to abiotic stresses such as high salinity. OsPQT3 knockout mutants (ospqt3) have increased resistance to oxidative and salt stress, as well as increased expression of OsGPX1, OsAPX1, and OsSOD1. E3 ubiquitin ligases play critical roles in various signalling processes in biotic and abiotic stress response by specifically recognizing targets, which are usually transcription factors (TFs), for degradation [175]. According to genetic data, OsMPK4 enhances salt response in an IPA1-dependent manner, whereas IPA1 negatively regulates salt tolerance in rice. When exposed to salt stress, OsMPK4 may interact with IPA1 and phosphorylate it at Thr180, causing IPA1 to degrade. Together, the salt-induced OsMPK4-IPA1 signal cascade modifies rice's response to salt stress and offers new insight into the development of salinity tolerant rice varieties [224].C/VIFs are proteinaceous inhibitors of vacuolar invertase (VI) and cell wall invertase (CWI). In the early stages of germination, the CRISPR/Cas c/vif1 mutants showed a small improvement in their tolerance to ABA as well as salinity in Arabidopsis ( [199]). Essentially, SAUR41s are cytosolic proteins. The CRISPR/Cas9 system's knockout of every SAUR41 subfamily member caused immature Arabidopsis seedlings to grow less quickly and become more vulnerable to salt stress [200].

5 Other relevant CRISPR gene targets to overcome salt stress

To survive in high-salt soils, plants develop a variety of physiological and biochemical mechanisms. Ion-homeostasis and compartmentalization, ion transport and uptake, biosynthesis of osmo-protectants and compatible solutes, antioxidant enzyme activation and synthesis of antioxidant compounds, and hormone modulation are some of the primary mechanisms [111]. CRISPR technology has also been used to manipulate these physiological and biochemical processes in order to develop salt resistance in plants. The following section discusses research advances in elucidating the use of CRISPR technology in modifying genes related to these mechanisms.

5.1 Osmo-protectants

The ACQOS locus is made up of four nucleotide-binding leucine-rich repeats (NLRs) that encode a protein of the toll-interleukin1 receptor-nucleotide-binding leucine-rich repeat class. In, Arabidopsis, salt sensitivities are found in ACQOS alleles. The knockout line of the ACQOS allele was established using the CRISPR/Cas9 system. Because the chlorophyll content was significantly lower in mutants compared to the wild type, chlorophyll measurements suggested that ACQOS silencing significantly affected salt stress tolerance [197].

A study was conducted to create mutant alleles of the drought and salt tolerance (DST) gene for a zinc finger protein in Indica rice cv. MTU1010. Two different gRNAs were used to target DST protein regions that may be involved in protein–protein interaction and successfully generated different mutant alleles of the DST gene. For phenotypic analysis, homozygous dst mutants with a 366 bp deletion between the two gRNAs were chosen. This 366 bp deletion resulted in the deletion of amino acid residues 184 to 305 in frame, giving rise to the mutant name dst184−305. The mutant had wider leaves with lower stomatal density, which improved leaf water retention under dehydration stress. In the seedling stage, the Cas9-free dst184-305 mutant showed moderate tolerance to osmotic stress and high tolerance to salt stress [176].

5.2 Hormonal responses

Plant hormones have long been recognized as crucial endogenous molecules that control how well plants tolerate or are susceptible to a variety of stresses, including salinity stress. Abscisic acid (ABA), auxin, cytokinins (CK), brassinosteroids (BRs), jasmonate (JA), gibberellin (GA), and ethylene (ET) are examples of plant hormones that have been investigated for their potential role in reducing salt stress in plants [225].

Auxin response factors (ARFs) play an important role in the regulation of auxin response genes in tomatoes. Under NaCl exposure, SlARF4 mutants were obtained using CRISPR/Cas9, and their loss of shoot fresh weight was half that of the wild type [194]. One of the negative regulators of tomato multi-stress responses have been identified as hybrid proline-rich proteins (HyPRPs), a subclass of putative plant cell wall glycoproteins. Multiplexed editing of SlHyPRP1 with CRISPR/Cas9 resulted in precise deletions of its functional motif(s), resulting in salt stress-tolerant events in cultivated tomato. Engineering the SlHyPRP1 gene by accurately removing its PRD, 8CM domain, or both resulted in higher overall survival than the wild type in medium containing 150 mM NaCl [193]. OsVDE, a lipocalin-like protein found in chloroplasts, promotes ABA biosynthesis while suppressing salt-stress tolerance in rice seedlings. The OsVDE mutant produced by the CRISPR/Cas9 system had greater stomatal closure and a higher ABA content than the wild type, resulting in less water loss from transpiration [172].

5.3 Membrane transporters

The significance of Na+ exclusion in protecting plants from salinity stress is widely acknowledged. Under salt stress, the ion-exchange activity of Na+ influx and efflux determine net Na+ accumulation in plant cells. Na+ influx is primarily mediated by ion channels such as the high-affinity K+ transporter HKT and non-selective cation channels (NSCC), while Na+ efflux is mediated by SOS1, a Na+/H+ antiporter. Under saline conditions, Na+ efflux from plant cells is an active process in the presence of elevated levels of external Na+. According to Zhang et al., [226], ZmHKT1 encodes a plasma membrane-localized Na+-selective transporter that is preferentially expressed in root steles in maize (including the parenchyma cells surrounding the xylem vessels). ZmHKT1 function loss via CRISPR-Cas9 technology increases xylem sap Na+ concentration and root-to-shoot Na+ delivery, indicating that ZmHKT1 promotes leaf Na+ exclusion and salt tolerance by withdrawing Na+ from the xylem sap. Another study has demonstrated the role of the sodium/hydrogen exchanger gene, GmNHX5, in salt tolerance in soybean. GmNHX5 is induced by salt treatment, and was found in the Golgi apparatus, distributed in new leaves and vascular. Whereas GmNHX5 deletion by CRISPR/Cas9 resulted in hairy roots that were less salt tolerant, overexpression of GmNHX5 had the reverse effect [171]. When exposed to salt stress the SlHAK20 transports Na+ and K+ and controls their equilibrium. A change in the coding sequence of SlHAK20 was revealed to be responsible for salt tolerance in tomato. Hypersensitivity to salt stress was caused by knockout mutations in the rice homologous genes and the tomato SlHAK20 gene [174]. The SlSOS1 controls Na+ and K+ homeostasis in tomato roots and shoots under salt stress conditions. Furthermore, the phenotype analysis revealed that wild type plants and slsos mutants clearly responded differently to salt stress, indicating that SlSOS1 is essential for tomato salt tolerance [196].

6 Advantage of CRISPR approach over breeding and transgenic technologies

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system is a novel tool in the field of gene editing that can be used to precisely and specifically edit a specific genomic region. This system is widely used, especially when investigating new gene functions, and it helps create mutant lines that are more resistant to abiotic stressors. Also, the CRISPR/Cas system's notable feature lies in its capacity for multiplex editing, enabling the mutation of multiple genes in a single transformation event. This capability streamlines the process, ultimately reducing the time and cost associated with creating new varieties. Furthermore, it is useful in the creation of salinity stress resistant varieties and strains with higher yield potential. When it comes to genetic modification, the CRISPR method has unambiguous benefits over conventional breeding and transgenic technologies. Following are the main highlights:

6.1 Precision and specificity

The high-cost-effectiveness, less tedious, precision and specificity make CRISPR better and widely used technology. Nevertheless, achieving optimal genome-editing efficiency with minimal or no off-targets remains a formidable challenge. The CRISPR/Cas system's defense mechanism, orthologues, applicability, and efficiency in editing genomes are all influenced by a number of factors. Numerous experimental improvements have been made to the CRISPR/Cas system since it was first used to edit genomes. For example, the GC content of designed sgRNA must range from > 30% to < 70%; truncating gRNA or adding extra guanines at its 5′end increases its specificity; replacing one of the nucleotides in the continuous stretch of four to six; avoiding constitutively higher expression levels of sgRNA and Cas9 to prevent off-targeting; selecting appropriate method to deliver CRISPR components and several others [227]. Despite the remarkable progress in this editing technology, there are still a lot of basic mechanical problems about how to acquire spacers and distinguish between different CRISPR subtypes that are self or non-self. For some subtypes, such type I-E and type II-A, the mechanism of crRNA synthesis and interference is quite well characterized. Further characterization of Type IV, V, and VI is necessary, nevertheless, as some of them exhibit mechanisms that differ from those of the conventional systems [228]. Furthermore, a multitude of subtypes with potentially novel mechanisms of action must be identified to improve the system's biotechnological application.

6.2 Speed and Efficiency

Modern gene editing techniques such as CRISPR/CAS9 have surfaced in recent years, replacing more traditional approaches such as natural selection, random mutagenesis, and classical breeding. This change is a result of the traditional methods' time-consuming nature, which frequently requires long time to produce individuals with the desired phenotype [229]. The DNA-binding mechanism of method differs from that of ZFN and TALEN. An easily constructed 20 base pair (bp) RNA guide sequence that binds to its DNA target by base-pairing allows Cas9 to be targeted to a specific genomic sequence. For instance, a recent review by [230] examined the application of various gene editing techniques in rice from 2013 to early 2018. Rice and other important crops like tobacco, sorghum, wheat, and Arabidopsis were among the many plants whose gene editing was transformed by CRISPR/Cas technology. The original documentation of the groundbreaking work in this field was done in 2013 by [231], Miao et al., [232] and Jiang W Z et al. [164]. Shan et al. [233] presented the design of two single-guide RNAs (sgRNAs) that specifically target the rice phytoene desaturase gene (OsPDS) in a seminal study. In transformed rice (Nipponbare) protoplasts, the experiment demonstrated an astounding 15% mutagenesis efficiency. The accuracy and efficiency of CRISPR methods for genetic modification and agricultural improvement have significantly improved as a result of this discovery. Two sgRNA expression plasmids, one specifically designed to target OsPDS and the other to target BETAINE ALDEHYDE DEHYDROGENASE 2 (OsBADH2), were bombarded into Nipponbare calli. According to the analysis, OsBADH2 mutations were found in 7 out of 98 transgenic plants (7.1%), while OsPDS mutations were found in 9 out of 96 independent transgenic plants (9.4%).

6.3 Multiplex editing capability

One particularly useful feature of the CRISPR/Cas system is its multiplex editing capability. It makes it possible for numerous genes to mutate simultaneously during a single transformation event, significantly cutting down on the time and expense involved in producing new varieties. In the case of the Nipponbare and Kitaake rice varieties, this characteristic has been effectively utilized to cause the simultaneous mutation of an entire gene family. For example, four members of the mitogen-activated protein kinase gene family are the targets of an eight-sgRNA set that has been painstakingly created. The two pairs are aimed at two different genomic locations within a gene locus, which are spaced 350–750 base pairs apart to make it easier to see excision in cells that have been edited. With the help of the endogenous tRNA-processing system, the synthesis of multiple sgRNAs is simplified using a single polycistronic gene, guaranteeing accurate cleavage at both ends of the tRNA precursor [234].

6.4 Transgene-free mutant plants

CRISPR/Cas offers a crucial advantage wherein mutant plants devoid of transgenes can be obtained in the initial generation. This can be accomplished through the segregation of the transgene. For instance, many reports of T1 transgene-free mutant plants have been made in the field of rice [235, 236]. A different method of obtaining transgene-free mutant plants is by DNA-free gene editing with ribonucleoprotein (RNP) complexes, which include Cas9 and sgRNAs. In plant cells, CRISPR/Cas9 reagents are expressed via RNA and RNPs in order to entirely prevent DNA integration [237, 238]. These techniques also reduce off-target mutations, which continue to be a significant issue with the integration of CRISPR/Cas9 [237, 239, 240]. However, most labs are unable to easily apply RNPs due to their delivery challenges [241, 242]. Currently, the most practical and effective method for producing transgene-free genome-edited plants is probably the use of RNA viruses to introduce CRISPR/Cas9-expressing RNA into plant cells [243]. However, limitations on the host range linked to certain viruses continue to be a significant barrier to this strategy's application. Currently, only in tobacco, CRISPR/Cas9 delivery via RNA viruses is applicable. To increase delivery efficiency, new delivery mechanisms for CRISPR/Cas9 RNA and RNPs must be developed. Additionally, more reliable screening systems must be established in order to distinguish transgene-free mutants from controls. These developments are desperately needed to encourage the use of CRISPR/Cas9 technology in agriculture.

7 Challenges and constraints in utilizing CRISPR/Cas technology in plant genome editing

To ensure future food security, the development of plant varieties with specific traits is imperative. The field of plant breeding has entered a new era thanks to gene editing technologies like CRISPR/Cas9 systems. Despite these advancements, several challenges persist in the use of CRISPR/Cas9-based gene editing in plants [244]. While there have been successful instances of homologous recombination (knock-in/gene replacement) using CRISPR/Cas in plants, the editing efficiency remains a significant hurdle [245, 246]. This underscores the ongoing journey toward achieving high-efficiency gene knock-in through CRISPR/Cas-mediated homologous recombination within the field of plant genetics [160]. The primary drawback of CRISPR/Cas9, when not meticulously designed and executed, is the occurrence of increased off-target effects [247, 248]. Also, several constraints exist regarding the application of CRISPR in plants. Firstly, direct targeting of specific genes (S genes) can lead to a fitness cost due to their linkage with other favorable genes, notably those controlling essential plant growth and development. Secondly, the prevalence of "off-target mutations" poses a challenge in refining the CRISPR system, particularly in the creation of transgene-free crops. Thirdly, concerns about safety and the implications for commercialization are intertwined with potential impacts on human health and other living organisms [249].

The possibility of unexpected off-target events when using CRISPR/Cas technology to edit genomes presents a serious challenge. A significant obstacle to the widespread use of gene editing technologies in plant breeding has been this worry. Nonetheless, this aspect of gene editing has been addressed and improved by the use of more recent nucleases, such as Cpf1, which has improved specificity over Cas9. For instance, [250] conducted a comparison of two nucleases, CRISPR/Cas9 and CRISPR/Cpf1, for gene editing in the indica variety IR64. Their findings suggest that both systems exhibit comparable efficiency in editing the EPIDERMAL PATTERNING FACTOR LIKE 9 (EPFL9) gene. This regulatory approach appears scientifically inconsistent, as it seeks to control systems that generate highly specific and singular genomic modifications, while leaving unregulated methods like chemical and radiation mutagenesis, which result in thousands of random mutations [251]. Moreover, despite the significant benefits offered by CRISPR technologies for enhancing crops, there are instances where these advancements may be restricted in certain countries due to governmental policies that classify gene-edited products as genetically modified organisms (GMOs). Without a universal, clear, and scalable regulatory system, CRISPR-edited crops could encounter the same challenges as GMOs. If these crops and their derived products are classified and regulated in the same way as GM crops, their future cultivation and public acceptance, particularly in the European Union (EU), are likely to be limited [252]. While CRISPR offers a high degree of precision, the scientific community remains concerned about off-target effects [253, 254]. Researchers have additional concerns about the environmental impact of CRISPR-edited crops and the difficulty of controlling them after they are released [255]. To date, different countries have regulated genome-edited crops differently due to differing definitions of genetic modifications and GMOs. The regulatory paradigm in the United States, known as the coordinated framework, distributes authority among the US Food and Drug Administration (FDA), the Environmental Protection Agency (EPA), and the Department of Agriculture (USDA). This framework relies on existing statutes for regulatory authority rather than a national biosafety law, as is common in other parts of the world [256]. The continued reliance on process-based definitions for regulatory oversight, along with the use of process-focused language in public discourse, hinders the development of appropriately nuanced approaches for regulating genome-edited crops. The importance of understanding the nature of the novel plant phenotype or trait in safety assessments is frequently overlooked. This complicates regulatory approvals for crops developed using both traditional and new plant breeding techniques. Without a greater emphasis on this aspect, the public may misunderstand genome editing, and regulators may feel compelled to evaluate these products using out-of-date biosafety guidelines. Fortunately, regulators are making progress in developing sensible and practical approaches to using genome editing for crop improvement. However, new product-based paradigms for regulating new breeding technologies will inevitably emerge.

8 Conclusion

We have given an overview of how plants react to salt conditions and discussed how CRISPR technologies may be used to create crop varieties that are more resilient to salinity stress. An exceptional opportunity to trace the flow of genetic information across CD processes during stress biology exists thanks to an integrated understanding of genomics, epigenetics, transcriptomics, and proteomics. The CD has been a valuable model of genetic information transfer, and our understanding of the precise mechanisms involved in that transfer is still evolving. For clarifying gene functions and adjusting plant response pathways against salinity stress, CRISPR technology is a godsend in this area. The growing CRISPR toolkit makes it possible to target every mechanism that controls plant characteristics through precise and effective genome engineering of CD components. However, caution should be exercised when designing sgRNAs, as off-targets are a significant limitation, and not every gene or region can be efficiently targeted due to a lack of highly specific sgRNAs in the desired location and off-target activity is extremely common. Simultaneously, to ensure the success of CRISPR edited crops, all other agronomic traits of interest, such as responses to abiotic stresses and yield, must be subjected to rigorous field trials. While revisiting the CD of molecular biology, several analogous linkages within the CD for salinity tolerance can be found, enabling plant researchers to develop new salinity tolerant cultivars in future.