1. Introduction
By the end of the year 2050, the world population is anticipated to reach up to 10 billion [
1]. In this situation, increasing food crop production by 60% over the coming decades is necessary to ensure global food security [
1,
2]. To sustainably increased food production, additional integration of all developed relevant techniques, such as genomics, genome editing (GE), artificial intelligence, and deep learning, will be necessary [
3,
4]. Crop modification methods have a long history and have been used ever since the first agricultural plants were domesticated. Since then, other new methods have been created and are being developed to boost crop production and economic value even more. Traditional crop breeding techniques in the 20th century either relied on naturally occurring mutations or on mutagenesis that was created artificially [
5]. Genetic research has traditionally focused on the identification and assessment of spontaneous mutations. Scientists were reliant on each other and showed that radiation or chemical treatment could increase the rate of mutagenesis [
6,
7]. Later approaches, suchas radiation and chemical mutagenesis, altered the genome at random sites by inserting transposon motifs that may be induced in some animals. However, a fundamental disadvantage of conventional breeding methods is the length of time needed to breed new varieties of any crops with the required agronomic characteristics. The duration of the growing season and the maturity level of the plants (particularly long-period growers, such as trees), as well as various stages of crossing, selection, and testing during the breeding process, all have an impact on this [
8]. The plant genome cannot be targeted using conventional techniques for chemical and physical mutagenesis or natural mutations. Using genetic engineering, better plants and animals may be developed more quickly [
5].
The first genetically modified (GM) crops were released for sale in 1996 [
9]. Generations of GM crops up to now have relied on the genome’s random insertion of new DNA sequences. The possibility that the inserted gene may affect or impede the activity of other crucial nearby genes has been raised as a concern regarding this approach. In addition, public anxiety regarding GM crops is increased when talking about the introduction of ‘alien’ genes from distantly related organisms, which is thought to be ‘unnatural’ despite mounting evidence to the contrary [
10,
11].
The creation and use of DNA-based markers at the turn of the twenty-first century has made it possible to reduce significantly the time needed to generate new lines and varieties of agricultural crops [
10,
11,
12,
13]. All these factors have greatly helped the development of focused GE methods [
14,
15,
16,
17]. In yeast and mice, the first targeted genetic alterations were created in the 1970s and 1980s [
6,
8]. This gene targeting was based on the homologous recombination process, which was extremely accurate.
RNA interference (RNAi) was one of the first GE technologies [
5,
18,
19]. Even though this technology has been successfully used in functional genomics and plant breeding [
20,
21,
22], it has several drawbacks, including the unlimited insertion site of an RNAi construction into the genome and partial gene function suppression [
5].
This is a marvelous time for genetics, due to advances in genetic analysis and genetic manipulation. Genome editing, the most recent crop-enhancement method, allows precise changes of the plant genome by deleting undesired genes or enabling genes to acquire new functions [
23]. Numerous crops’ genomes have been sequenced, and improvements in genome-editing techniques have made it possible to breed for desired features. To sustainably increase food production, additional integration of all developed relevant techniques, such as genomics, genome editing (GE), artificial intelligence, and deep learning, is necessary [
24].
Advanced biotechnological methods are made possible by genome-editing tools, allowing for precise and effective targeted modification of an organism’s genome. Several novel tools for genome or gene editing are available to enable researchers to modify genomic sequences precisely [
25]. These techniques facilitate novel insights into the functional genomics of an organism and enable us to alter the regulation of gene expression patterns in a pre-determined region. Because of accurate DNA manipulation, genome-editing technologies, for instance, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated systems), TALENs (transcription activator-like effector nucleases), CRISPR/Cas12a (Cpf1, CRISPR from
Prevotella and
Francisella1), and Cas9-derived DNA base editors, provide unprecedented advancements in genome engineering. As a result, this technology is a powerful tool that can be employed to secure the global food supply [
26].
Genome editing was first proposed by Capecchi [
27] in the 1980s. This method allows for the removal, modification, or addition of genetic material at specified genomic locations. Even though current GE technologies are substantially more accurate than traditional mutagenesis [
28,
29], the biggest barrier here is still the legitimacy of GE crops. Assessing the biosafety of such crops is a unique difficulty because it is impossible to predict the effects of single base alterations following the application of ODM and BEs [
30,
31].
The primary elements that affect plant growth and reduce agricultural productivity are biotic stressors [
32,
33] such as disease and insect pests, along with abiotic stressors [
13] including cold, drought, and saline–alkali stress (
Figure 1). Many crop plants that can withstand abiotic stress have previously been created via traditional marker-assisted breeding. However, due to extensive screening [
34,
35] and backcrossing procedures, it takes this tactic about a decade to generate abiotic stress-resilient crops effectively [
36]. Although genetically modified, stress-tolerant plants have disclosed encouraging results, several barriers still stand in the way of their widespread commercialization. In many ways, crops with genome editing differ from genetically engineered species [
37]. Considering this, genome editing seems to be a sophisticated strategy to create crops that are resistant to different abiotic stress in the future, because it allows precise manipulation of different gene loci in comparably less time, which lowers the cost of crop-improvement programmes [
38]. Gene-editing technology based on CRISPR/Cas might successfully target complex quantitative genes linked either directly or indirectly to abiotic stressors. The use of CRISPR-Castechnology has been linked in recent years to the establishment of disease resistance in plants by modifying gene regulation [
39,
40,
41,
42]. Currently, CRISPR/Cas-based genome editing has been efficaciously utilized to investigate tolerance against multiple abiotic stresses, including heat, drought, salt, and nutritional values in several critical agricultural plants [
43,
44]. In this review article, we summarize the most likely uses of the CRISPR/Cas9-mediated genome editing technique in crop plants for dealing with diverse abiotic stresses such as heat, drought, salinity, cold, herbicide etc., and we predict the tools for future advancements in the creation of crop varieties that can withstand stresses.
3. Genome Editing Related to Abiotic Stresses
Abiotic stresses that impact plant growth and development, such as salt, drought, extremely high temperatures, cold, and heavy metals, can reduce agricultural production by approximately 50% [
124].Numerous biochemical, morphological, and physiological factors important for plant development are influenced by stress. Stresses from the environment can modify how plants behave as they develop. Most changes in plant growth and development caused by different abiotic stresses are associated with poorer yields [
13]. By 2050, the rapid growth in the human population is predicted to reach 9.7 billion. The global temperature is also set to increase significantly. As plant scientists, it is hard for us to manage the food requirements of the increasing population. However, we own the capability to develop climate-flexible crop varieties that can flourish under such challenging circumstances. These varieties must be maintained in ruthless climatic conditions such as heat, drought, heavy metals, cold, or flood stresses. This requires a continuous search for newer and diverse germplasm [
125,
126], which was traditionally performed either entirely through development of natural variations [
127,
128] or by selective breeding [
129,
130]. Another possibility is the construction of mutant populations that are evaluated to hunt for new resources among variations that might be novel valuable mutations that in turn are included in breeding programmes. Modern genome-editing system tools such as CRISPR facilitate the user to commence desirable genomic modifications accurately, illustrating great promise as a tool for producing novel climate-resistant plants [
131]. In over 20 agronomically important crops, CRISPR/Cas mediated gene editing is widely utilized and accepted for crop improvement against different abiotic stresses [
79].
Ordinarily, plants are equipped with numerous defense schemes against abiotic stresses. Among numerous defense mechanisms of abiotic stresses, the five broad-spectrum protections are regulated utilized in a complicated managing network consisting of numerous mediators and gene regulatory constituents in response to abiotic stresses [
132]. During the procedure, stress hormones, particularly nitrogen oxides (NO), abscisic acid (ABA), polyamines (PAs), calcium ions (Ca
2+), hydrogen sulfide (H
2S), reactive oxygen species (ROS), and phytochrome B (PHYB), interact with others, either synergistically or antagonistically. The transcription factors (TFs) could alter the expression of genes and enzyme activity in a regulatory way, triggering a suitable reaction. The regulatory constituents open a lot of potential for developing multiple stress tolerance/resistance. Five main plant defenses to abiotic stresses are ROS scavengers, molecular chaperones, cuticle as the outer shield, oxylipin precursors, and osmoprotectants, along with unsaturated fatty acids, and compatible solutes [
132].
3.1. Drought Stress
Drought is becoming a challenge to sustainable agriculture due to the consequences of climate change, including erratic rainfall patterns and rising temperatures in many regions of the world. The greatest danger to global food security is drought stress, which is the primary factor in the catastrophic loss of agricultural production and productivity [
133]. Drought alone can reduce yield by 50–70% in different crops [
134]. For example, 40% yield losses due to drought stress have been reported in maize [
35,
135], 50% in rice [
136], 21% in wheat [
126,
135], 27–40% in chickpea [
125,
137], 68% in cowpea [
138] and 42% in soybean [
34,
139]. After the discovery of genome editing, efforts are being planned to alter the genes involved in pathways enabling drought tolerance, in order to increase farmers’ acceptance of crops using these technologies. In recent years, in-depth research has helped to adapt and overcome drought stress using CRISPR-Cas9 technology (
Table 3).
In many crop plants, H
2O
2 and abscisic acid (ABA) are frequently produced in situations of salinity or drought stress. The discovery was reported of ABA-induced transcription repressors (AITRs) as a novel transcription factor family that plays a significant role as feedback regulators of ABA signaling. Alternation in the expression of AITR genes resulted in abiotic stress tolerance, including drought and salinity in
Arabidopsis [
140,
141]. A CRISPR/Cas9-induced mutation in the
Arabidopsis OST2 structural gene exhibited drought resistance [
142]. Another study found that knockout of Arabidopsis plants’ genemiR169athrough CRISPR/Cas9 led to significantly improved drought tolerance [
143]. Similarly, Arabidopsis’ drought tolerance increased after the vacuolar H+-pyrophosphate (AVP1) regulating gene was expressed using CRISPR/Cas9 [
144]. Similar results were shown when the abscisic acid-responsive element binding gene (AREB1) was activated in Arabidopsis through CRISPR/Cas9a [
145]. Recently, drought tolerance in
Arabidopsis thaliana was demonstrated via the CRISPR/Cas9 gene silencing of the trehalose (TRE1) gene [
146].
Numerous studies have documented how CRISPR confers drought resistance in many plants. For instance, it has been demonstrated that increasing rice’s ability to withstand drought can be attained by reducing the expression of the regulatory genes DERF1, PMS3, MSH1, MYB5, and SPP [
147]. In rice plants, drought stress tolerance increased after OsERA1 was modified using CRISPR/Cas9 [
148]. CRISPR/Cas9 has been employed to improve drought resistance in rice by knocking out the SRL1, SRL2, and ERA1 genes [
148,
149]. A CRISPR/Cas9-created ospyl9 mutant might increase rice yield and drought tolerance [
150]. Indica mega rice cultivar MTU1010 with broader leaves, a decreased stomatal density, and improved leaf water retention under drought stress was developed using CRISPR/Cas9 to modify the
OsDST gene [
151]. The
OsOREB1,
OsRab21,
OsRab16b,
OsLEA3,
OsbZIP23,
OsSLAC1, and
OsSLAC7 genes, which act downstream of SAPK2, were modulated in expression in the loss-of-function sapk2 mutant of rice plants developed using CRISPR/Cas, increasing their tolerance to drought stress [
131].
Two genes,
RVE7 and
4CL, have been found to be associated with drought tolerance in chickpeas. The first report of CRISPR/Cas9-mediatedediting of the chickpea protoplast was made by Badhan et al. [
152]. They described knockouts of the genes
4CL and
RVE7, which are linked to pathways for drought tolerance. That study established a framework for potential future chickpea-genome-editing approaches [
153]. Another gene, namely
ARGOS8, responding to drought stress has been altered through genome editing. The expression of the
ARGOS8 gene increased as a result of negative regulators of ethylene signaling pathways, providing drought tolerance [
154,
155]. To increase the production of maize under drought stress under field conditions, the GOS2 promoter region was replaced with an
ARGOS8 promoter sequence using the CRISPR/Cas system [
156].
CRISPR/Cas9 altered the
GID1 gene in tomato plants, which exhibit high leaf water content under drought conditions [
157]. Additionally,
SlLBD40 gene mutation caused by CRISPR/Cas9 significantly improved drought tolerance in tomato [
158]. Furthermore, use of the CRISPR/Cas technique to alter mitogen-activated protein kinases (MAPKs) revealed SlMAPK3 to be a drought stress modulator [
159]. Knockout of the
SINPR1 gene resulted in increased drought tolerance and down-regulation of drought-related genes [
160].
Drought resistance of wheat was improved by CRISPR/Cas editing of wheat
TaDREB2 and
TaERF3 [
89]. In wheat, a multiplex CRISPR/Cas9 assay was used to alter the
SAL1 gene, a negative regulator of drought tolerance, to increase drought tolerance at the seedling stage [
161]. CRISPR/Cas genome editing of the HB12 gene can increase cotton’s resistance to drought [
162]. CRISPR/Cas9 was used to modify the
BnaA6.RGA gene in oil seed crops, which significantly improved rapeseed’s ability to withstand drought [
163].
3.2. Heat/Temperature Stress
Plants have a preferred temperature, any rise or fall in that temperature can significantly impede their development and productivity. The third most important abiotic factor is heating stress, which may decrease crop production considerably. For instance, every 1 °C augmentation in atmospheric temperature diminishes wheat yield by 6%, rice yield by 10–20%, and corn yield by 21–31% [
164,
165,
166]. Significant yield losses were caused by high heat stress, which is now recognized as a severe problem that will simply become worse in the future. All phases of plant growth, from germination to harvest, are severely harmed by heat stress [
167,
168]. Heat stress not only increases plant mortality rates but also reduces plant quality [
169,
170].
In severe cases, a bad alteration in temperature results in plant mortality because plants are more susceptible to temperature changes. The ideal temperature would normally be better for crop growth and development; conditions below and above the optimum temperature have a harmful effect on productivity. For every 10 °C rise, followed by 20 °C and 30 °C, mostbiochemical and enzymatic procedures double in speed [
171]. Abiotic stressors, predominantly high and low heat, have a harmful effect on the premature stage of the male gametophyte in a range of agricultural crops, including maize, rice, barley, wheat, sorghum, and chickpea [
172]. Due to temperature stress, the functions of tapetal cells are diminishedduring the reproductive growth period, and the anther is dysplastic. Pollen discharge is insufficient and indehiscence happens as a result of increased heat preventing pollen grains from escalating. Plants have developed precise physiological and chemical reactions to manage temperature stress [
173].
The presence of genes that are responsive to heat stress, signal transduction, and the synthesis of metabolites are only a few of the complex molecular systems that plants activate in response to heat stress. Different temperature-stress-related genes have been identified and characterized to improve plants’ ability to withstand heat as a result of developments in structural and functional genomics technologies in plants. The heat stress reaction, which is connected to the accumulation of ROS, is mediated by the heat shock transcription factors (HSFs) and the heat shock proteins (HSPs) [
174].Therefore, by enhancing plants’ ability to resist ROS components, temperature stress tolerance can be improved [
175]. This indicated that higher tolerance might increase the antioxidant properties of crops. Plant temperature tolerance was significantly increased via metabolite production and temperature-induced gene expression. To explore the molecular processes associated with temperature stress and improve plant heat tolerance, CRISPR-Cas9 is a cutting-edge technology among all genome-editing techniques [
176] (
Table 3).
A cultivable HS-inducible rice mutant was created using CRISPR/Cas9 technology [
177]. The orthologs of mitogen-activated protein kinase 3 and agamous-like 6 were modified using CRISPR to increase tomato sensitivity to heat stress, whereas ADP-ribosylation factor 4 enhanced tomato sensitivity to salinity shocks. According to Bouzroud et al. [
178], these CRISPR-edited mutant plants had improved agronomic characteristics and were resilient to abiotic stresses. As a component for heat tolerance, BRZ1 positively regulates the formation of ROS in the tomato apoplastic area. This was confirmed by the CRISPR-Cas9-based bzr1 mutants, which showed reduced temperature tolerance and respiratory burst oxidase homolog 1 (RBOH1) with diminished hydrogen peroxide generation in the apoplast [
179]. In comparison to wild-type crops, the development of CRISPR/Cas-mediated heat-stress-sensitive albino 1 (HSA1) mutants of tomato showed greater sensitivity to temperature stress [
180].
The thermosensitive genic male sterile gene was altered by CRISPR in maize to promote thermo susceptible male-sterile plants [
181]. In lettuce, knockouts of NCED4, a crucial regulating enzyme in abscisic acid production, allowed the seeds to germinate at a higher temperature. As a result, LsNCED4 mutants may have commercial significance in manufacturing environments with high temperatures [
182]. In order to make a plant more resistant to heat, the hsps gene, which increases osmolyte levels and prevents cell protein damage, can be overexpressed [
183]. The protein kinase SAPK6 and the transcription factor OsbZIP46CA1 in rice also increase the capacity for responding to heat stress [
184].
3.3. Cold Stress
Cold stress, which includes chilling (20 °C) and freezing (0 °C) temperatures, hinders plant growth and development and severely limits plant geographic expansion and agricultural productivity [
185]. Plants are directly inhibited from responding metabolically to low temperatures, which results in osmotic stress, oxidative stress, and other types of stress. Due to mechanical damage and metabolic dysfunction caused by extreme cold temperatures, plant growth and development are halted [
186]. The physiological, biochemical, and molecular behavior of plants during their growth and expansion is adversely affected by cold stressors. The photosynthetic capacity and crop anatomy are brutally impacted by cold exposure, especially throughout the winter [
187,
188].Cold stress during the seedling stage may cause impaired germination and emergence. Long-term exposure impairs source–sink relationships, growth, nutrient localization, and leaf chlorosis [
189]. Membrane formation, which amplifies other cold-stress-related downstream processes, is the main consequence of cold stress on crops [
190]. In-generic or inter-specific hybridization has been successful in boosting the cold tolerance of significant crops using conventional breeding methods. For creating non-transgenic genome-edited crops to combat climate change and ensure future food security, CRISPR/Cas9 is a clever and practical approach [
191,
192] (
Table 4).
To increase the plant’s resistance to cold, genome editing is employed to target a few of the depressant regulator transcription factors in rice. A transcription factor called OsMYB30 attaches to the amylase gene promoter and negatively affects cold tolerance. According to Lv et al. [
193], under conditions of cold stress, OsMYB30 forms a compound with OsJAZ9 and slows down the expression of the amylase gene, which may contribute to increasing cold sensitivity by causing maltose buildup and starch breakdown. In order to determine the specific function of the TIFY1a, TIFY1b, and Ann3 genes in rice’s ability to withstand cold stress, CRISPR/Cas9 technology has also been applied to these genes. The mutant outperformed the natural variation in terms of yield, temperature tolerance, and amount of germination prior to harvest [
194]. Using CRISPR base editing, suppression of photosynthetic genes in rice plants under cold stress has been shown to cause the white-striped leaves phenotype in the white stripe leaf 5 (wsl5) mutant line [
195,
196].
PRPs are proline-rich proteins that not only aid in dealing with low temperatures but also reduce nutrient loss, boost antioxidant activity, and aid in the production of chlorophyll. Rice capacity for cold tolerance was improved by the CRISPR/Cas9 deletion of OsPRP1, which encodes a proline-rich protein [
197]. In a recent work using CRISPR/Cas9, three rice genes, viz., OsPIN5b, GS3, and OsMYB30were altered to increase spike length, grain size, and resilience to cold stress [
198]. The CRISPR/Cas9 technology altered the G-complex-related genes i.e., OsRGA1, OsGS3, OsDEP1, and OsPXLG4 to make rice more resistant to chilling stress [
199].Because tomato plants are prone to chilling stress, their fruits are more vulnerable to damage from the cold. C-repeat binding factor 1 (CBF1) was shown using CRISPR-Cas9-based cbf1 mutants to protect the tomato plant next to it from cold/chilling damage and decrease electrolyte leakage [
200]. These plants also demonstrated excellent addition of hydrogen peroxide and indole acetic acid, resulting in tomato plants tolerant of chilling stress.
Table 3.
List of reported targeted gene(s) via CRISPR/Cas9 technology in different plant species for development of tolerant genotypes against drought and heat stresses.
Table 3.
List of reported targeted gene(s) via CRISPR/Cas9 technology in different plant species for development of tolerant genotypes against drought and heat stresses.
Crops | Gene | Trait | Technique | References |
---|
Rice | OsDERF1 | Drought | CRISPR/Cas9 | [147] |
Rice | SRL1, SRL2 | Drought | CRISPR/Cas9 | [149] |
Rice | OsAAA-1, OsAAA-2 | Drought | CRISPR/Cas9 | [201] |
Rice | OsNAC006 (transcription factor) | Drought and heat sensitivity | CRISPR/Cas9 | [202] |
Rice | OsAOX1a | Drought resistance | CRISPR/Cas9 | [147] |
Rice | OsDST | Drought and salinity | CRISPR/Cas9 | [151] |
Rice | OsERA1, OsPYL9 | Drought | CRISPR/Cas9 | [148,150] |
Rice | SAPK2 | Tolerance to salinity and drought | CRISPR/Cas9 | [131] |
Rice | OsPMS3 | Photoperiod-sensitive male-sterile | CRISPR/Cas9 | [147] |
Rice | Csa | Photosensitive-genic male-sterile | CRISPR/Cas9 | [203,204] |
Rice | TMS5 | Thermo-sensitive genic male-sterile | CRISPR/Cas9 | [205] |
Rice | OsNAC14 | Drought tolerance | CRISPR/Cas9 | [206] |
Rice | OsPUB67 | Drought tolerance | CRISPR/Cas9 | [207] |
Wheat | TaDREB2, TaERF3 | Tolerance to drought | CRISPR/Cas9 | [89] |
Maize | ZmARGOS8 | Drought | CRISPR/Cas9 | [156] |
Maize | ZmTMS5 | Creation of thermosensitive maize lines | CRISPR/Cas9 | [181] |
Mustard | BnaA6.RGA | Drought tolerance | CRISPR/Cas9 | [163] |
Soybean | Drb2a, Drb2b | Tolerance to drought and salinity stress | CRISPR/Cas9 | [208] |
Soybean | GmMYB118 | Drought tolerance | CRISPR/Cas9 | [209] |
Chickpea | 4CL, RVE7 | Drought tolerance | CRISPR/Cas9 | [152] |
Tomato | SIMAPK3 and SlNPR1 | Drought | CRISPR/Cas9 | [159,160] |
Tomato | SlARF4 | Drought | CRISPR/Cas9 | [140] |
Tomato | SIAGL6 | Heat stress | CRISPR/Cas9 | [210] |
Table 4.
List of reported targeted gene(s) via CRISPR/Cas9 technology in different plant species for development of tolerant genotypes against cold stresses.
Table 4.
List of reported targeted gene(s) via CRISPR/Cas9 technology in different plant species for development of tolerant genotypes against cold stresses.
Crops | Gene | Trait | Technique | References |
---|
Rice | OsMYB30 | Cold tolerance | CRISPR/Cas9 | [198] |
Rice | OsAnn3 | Cold tolerance | CRISPR/Cas9 | [211] |
Rice | OsAnn5 | Cold tolerance | CRISPR/Cas9 | [211] |
Rice | OsPRP1 | Cold tolerance | CRISPR/Cas9 | [212] |
Tomato | SlCBF1 | Cold tolerance | CRISPR/Cas9 | [200] |
Arabidopsis thaliana | AtCBF1, AtCBF2 | Cold tolerance | CRISPR/Cas9 | [213] |
3.4. Salinity Stress
Owing to the negative consequences of climate change, salinity stress has recently become much worse [
214]. Salinity stress is the second most severe abiotic danger that affects fertile lands as well as crop productivity [
215]. According to Morton et al. [
216] and Van Zelm et al. [
217], severe salts have an impact on about one-fifth of the irrigated agricultural area. Lack of good irrigation water, a changing climate, and excessive use of chemicals such as fertilizers and pesticides prolong the process of adding more land to the salinity stress zone. According to estimates made by Jamil et al. [
218], 50% of cultivable lands will be saline by 2050 due to the overuse of chemicals including fertilizers and pesticides. One of the most important and harmful factors that has a negative impact on soil quality and agricultural output is salt stress. When too many soluble salts accumulate in the crop root zone, it causes salinization of the soil because roots are unable to absorb water. Thus, osmotic stress and nutritional imbalance in plants have a negative impact on their morphology, biochemistry, and biomass, which ultimately causes irreparable plant damage [
219,
220,
221].
Reactive oxygen species (ROS) are intensified by salt stress, which has a detrimental effect on crops’ cellular and metabolic processes [
222,
223]. Lipid peroxidation, which causes membrane deterioration as well as protein and DNA damage, is a harmful effect of ROS [
224]. By diminishing chlorophyll content and stomatal conductance, salt stress hinders the development of the photosystem II and the transpiratory apparatus [
225]. Additionally, it decreases the water potential of the soil and leaves, which lowers plant turgor pressure by affecting water relations and causing osmotic stress [
226]. Plants suffer from decreased leaf area, lower photosynthetic rate, poor seed germination, decreased biomass production, and crop yield as a result [
227,
228,
229]. Salinity tolerance is the ability of a plant to maintain the equilibrium of biomass and/or output under conditions of salt stress. In order to tolerate salt, plants have several molecular and physiological mechanisms [
230].
Genome editing has the capacity to improve crops; there are yet few studies on its effective application in breeding plants that can withstand saline stress (
Table 5). In one such work, rice was modified to impart salt stress tolerance by editing the
OsRR22 gene, which encodes for a transcription factor (TF) involved in the control of signaling and the metabolism of cytokinins in plants [
231,
232]. Using CRISPR/Cas9 technology, the
OsRR22 gene was altered, and two homologous T
2 generations revealed improved salt tolerance with no discernible difference between the modified and wild-type lines [
232]. Using CRISPR/Cas9 technology, the paraquat tolerance-3 mutations (
OsPQT3) gave rice a high level of salt tolerance [
233]. The function of
OsmiR535 in salt stress tolerance was investigated using genome-editing techniques, and it was proposed that
OsmiR535 might be knocked out using CRISPR/Cas9 to enhance salinity tolerance in rice. Additionally, a homozygous 5bp deletion in the
OsmiR535 coding region might be a valid target for raising rice’s salt tolerance [
234]. Furthermore, some other genes increase the ability of rice to tolerate salt, using CRISPR/Cas9 technology by eliminating the
OsbHLH024 gene and increasing the expression of the ion transporter genes including
OsHKT1;3,
OsHAK7, and
OsSOS1 [
235]. When the rice
OsRAV2 gene was altered using CRISPR-Cas, the rice plants were able to survive under high salt conditions [
236].
Improvements in salt stress tolerance were seen in tomatoes after changes were made to the 8CM and PRD domains of the hybrid proline-rich protein1 (HyPRP1) [
247]. Additionally, the capability of crops to tolerate salt stress may be significantly increased by employing CRISPR/Cas9 technology to eliminate the
OsDST genes for rice [
151],
OsNAC041 [
238], and HvITPK1 [
246] for barley.
3.5. Heavy Metals Stress
An important issue for sustainable agricultural development is heavy metals, which seriously impair plant growth and productivity [
249]. Heavy metals (HMs) including Mn, Cu, Ni, Co, Cd, Fe, Zn, and Hg, among others, have accumulated in soils as a result of various human activities such the application of fertilizer, incorrect disposal of industrial waste, and unauthorized sewage disposal [
250,
251], or the hasty disposal of vehicle waste. They are either collected on the soil surface or leached from the soil into the groundwater [
252,
253]. Additionally, heavy metals cause oxidative stress by promoting the generation of hydroxyl radicals (OH), superoxide radicals, and hydrogen peroxide (H
2O
2) [
250,
254]. Plant physio-morphological activities are hampered by the accumulation of HMs, especially in the roots where they are blocked by Casparian strips or trapped by root cell walls, which eventually reduces crop output [
255]. When consumed, heavy metals accumulated in plants canseriously impair human health [
256].
To combat heavy metal stress in plants, CRISPR-Cas9-induced plant mutants may prove useful (
Table 6). In contrast to WT Co10 plants, the oxp1/CRISPR mutant of Arabidopsis plants exhibits resistance to Cd, indicating an increased capacity for heavy metal detoxification in mutant crops [
257]. Accordingly, study showed how indel mutations using gene-editing techniques could provide tolerance to heavy metals and xenobiotics in plants [
257]. Increased plant tolerance to heavy metals is influenced by a variety of genes [
258]. Several transporter genes in rice, including OsLCT1 and OsNramp5, are implicated in Cd absorption by the roots [
259]. The amount of Cd in rice has been reduced by CRISPR/Cas9-enabled gene-expression manipulation. Rice grains with OsNRAMP1 knocked out by CRISPR/Cas9 have decreased levels of Cd and lead (Pb) [
260,
261]. Eliminating an R2R3 MYB transcription factor called OsARM1 using CRISPR/Cas9 prevents rice from absorbing and transporting arsenic [
262].Cesium (Cs+) absorption and translocation in rice are regulated by the
OsHAK1 gene. Using the CRISPR-Cas9 technique, the cesium permeable potassium transporter
OsHAK1 was turned inactive [
263].
3.6. Herbicide Stress
In order to increase crop productivity, there is a need to manage weed growth with application of herbicides. Herbicides destroy non-target plants while also causing stress to the target plants and weed plants by interfering with or changing their metabolic processes. They also leave soil residues that are hazardous to the environment [
264,
265].The morphological, physiological, and biochemical traits of agricultural plants have been negatively impacted by the inappropriate application of herbicides. Herbicide toxicity reduces photosynthetic activity, which has a detrimental impact on the ability of crop plants to produce yield. One of the main goals for raising agricultural productivity is the development of herbicide tolerance in crop plants. To improve herbicide resistance in plants, genome editing including ZFNs, TALENs, and CRISPR/Cas technologies is an excellent tool (
Table 6).
Leucine, isoleucine, and valine are branched amino acids whose biosynthesis is catalyzed by the enzyme acetolactate synthase, which is encoded by the
ACETOLACTATE SYNTHASE (
ALS) gene [
266,
267]. It is a potential target of many herbicide improvement programmes. The recombination of acetolactate synthase using CRISPR/Cas9 produces herbicide resistance in rice [
268] and in watermelons [
269]. Additionally, using the same strategy and emphasizing the
ALS1 and
ALS2 genes, herbicide-resistant maize plants were produced [
270]. CRISPR-based editing in the
OsALS1 gene has been used to introduce herbicide tolerance characteristics into rice [
271,
272]. Glyphosate is one of the most imperative and quickly adopted herbicides for function in resistant crops such as soybean, maize, sugar beet, and chili pepper. The advancement of glyphosate-resistant plants requires changes in the machinery of some genes [
203]. 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme is implicated in the formation of aromatic compounds in crops with the transfer of phosphoenolpyruvate (PEP) enzyme for activating the reaction [
203,
273]. Glyphosate hinders the act of the EPSPS enzyme by inhibiting the add-on of glyphosate to the PEP enzyme binding sites, eventually blocking the formation of aromatic products and causing crop death [
203]. The endogenous EPSPS gene of rice was targeted with CRISPR/Cas9 to produce site-specific gene incorporation and substitution, which were fully transferred to the next generation with crops 100% resistant to the glyphosate [
203]. CRISPR/Cas9 was also utilized toproduce a mutation in the promoter of the EPSPS gene of chili to state this gene beneath the action of glyphosate [
274]. The resulting crops were reasonably resistant to glyphosate, and additional studies advised that selecting a diverse promoter may assist in the development of entirely resistant chili [
274].The modified genotypes of rice and flax now have enhanced tolerance to glyphosate as a result of the CRISPR/Cas9 change of two nucleic acid residues in the binding site of glyphosate–EPSPS [
91,
203]. Recently, herbicide resistance was developed in tomato plants by CRISPR-Cas9-based targeted mutations in EPSPS, PDS (phytoene desaturase), and ALS [
92].
Table 6.
List of reported targeted gene(s) via CRISPR/Cas9 technology in different plant species for tailoring herbicide and metal stress tolerance.
Table 6.
List of reported targeted gene(s) via CRISPR/Cas9 technology in different plant species for tailoring herbicide and metal stress tolerance.
Crops | Gene | Trait | Technique | References |
---|
Rice | C287T | Herbicide resistance | CRISPR/Cas9 | [274] |
Rice | BEL | Herbicide resistance | CRISPR/Cas9 | [71] |
Rice | OsALS1 | Herbicide tolerance | CRISPR/Cas9 | [271] |
Rice | EPSPS | Herbicide resistance | CRISPR/Cas9 | [203] |
Rice | SF3B1 | Herbicide resistance | CRISPR/Cas9 | [72] |
Wheat | ALS | Herbicide resistance | CRISPR/Cas9 | [275,276] |
Maize | ALS1 and ALS2 | Herbicide resistance | CRISPR/Cas9 | [270] |
Maize | MS26 | Herbicide resistance | CRISPR/Cas9 | [270] |
Soybean | ALS1 | Resistant to Chlorsulfuron | CRISPR/Cas9 | [277] |
Tomato | ALS | Resistant to Chlorsulfuron | CRISPR/Cas9 | [278] |
Tomato | SlEPSPS | Herbicide resistance | CRISPR/Cas9 | [92] |
Tomato | SlALS1, SlALS2 | Herbicide resistance | CRISPR/Cas9 | [92] |
Tomato | Slpds1 | Herbicide resistance | CRISPR/Cas9 | [92] |
Rice | OsTubA2 | Base editing | CRISPR/Cas9 | [279] |
Rice | OsHAK1 | Low cesium accumulation | CRISPR/Cas9 | [263] |
Rice | OsPRX2 | Potassium deficiency tolerance | CRISPR/Cas9 | [280] |
Rice | OsARM1 | Increase tolerance to higharsenic | CRISPR/Cas9 | [260] |
Rice | OsLCT1 | Less cadmium accumulation | CRISPR/Cas9 | [259] |
4. Conclusions and Prospects
Plants serve as sources of food, fiber, medicine, biofuels, and other goods. Farmers need new, superior cultivars in order to increase crop output and feed both the nation and the world. Plant breeders need a variety of tools for this purpose, including genomics and marker-assisted molecular breeding. Scientists can now implant desired traits more precisely and faster than in the past. Meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR) system are genome-editing tools that have been used with greater accuracy and efficiency than conventional breeding to enhance the quality of staple, oilseed, and horticultural crops. Today, there are several successful cases of “genome editing.” In order to edit genes accurately in the genomes of model and crop plants as well as a range of other organisms, genome editing employs designed nucleases as potent tools that target certain DNA sequences. A study of the literature on transcriptomics, biotechnology, genomics, and phonemics has shown that this novel approach to crop development is effective. CRISPR/Cas9-based genome editing is a genuinely innovative strategy. With genome editing, crops can effectively incorporate a variety of genetic traits. When these precise and powerful methods are applied to expedite plant breeding, they create certain outcomes. In order to accomplish a second Green Revolution and meet the escalating food demands of a quickly growing global population under constantly changing climatic conditions, plant breeding will advance with the help of this multidisciplinary approach. By overcoming the limitations of current transgenic techniques, genome-editing technology ushers in a new era of improved plant genetics. This information may be proved useful to plant breeders and researchers in their thorough evaluation of the use of various gene-editing tools to improve crops by focusing on the targeted gene. We believe that CRISPR/Cas9 technology islikely to bridge the GMO and societal divide in upcoming days.