*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 [219–221].

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 [227–229]. 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 T2 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].

**Table 5.** List of reported targeted gene(s) via CRISPR/Cas9 technology in different plant species for developing salinity tolerance.


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 (H2O2) [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.** *Cont.*

#### **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. **Author Contributions:** Writing—original draft preparation, R.K.Y., S.T., N.T., R.A., S.C., P.N.T. and D.K.P.; writing—review and editing, M.K.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
