Many important crop traits can be improved with a single base change in the genes and do not require DSBs and donor DNA templates for HDR. Single-base editing in such situations cannot be accomplished with the knock-in/out approach based on regular CRISPR-Cas system. Studies on agronomic traits suggest that numerous such traits are resolute by changes in the single bases of genes (Li et al., 2017). The CRISPR/Cas9 system has limitations and cannot be used to carry out gene base conversion. Therefore, it is the most suitable for knock-out or knock-in genes in the genome. Considering these restrictions, it is vital to find an accurate and robust method for editing the crop plant genomes.

A novel editing strategy that serves this purpose is base editing (Veillet et al., 2019), which accurately brings nucleotide changes in the absence of DSBs (Yin et al., 2017; Davies, 2019). The base editing pipeline still relies on the gRNA-guided target finding in the genome, however, involving the inactive CRISPR–Cas9 nuclease (which cannot make DSBs) fused to the deaminase enzyme (cytosine or adenosine) component that manipulates the nucleotide conversion.

A newly evolved cytidine base-editing tool proposes a valuable substitute (Komor et al., 2016). In wheat, acetolactate synthase (ALS) is an ideal herbicide tolerance target gene for base editing that can contain point mutations conferring adequate herbicide tolerance with a minor consequence to plant productivity(Yu and Powles, 2014). This is why mutations in ALS genes have been obtained through cytidine base editing in some diploid plant species, including Imidazolinone tolerance in rice (Shimatani et al., 2017)and tribenuron tolerance in Arabidopsis (Chen et al., 2010).

Compared to HDR-dependent CRISPR/Cas9, prime editing (PE) is homology-directed repair (HDR)-independent gene-editing technology that utilizes nCas9 attached to an engineered reverse transcriptase. At the same time, template RNA is linked to sgRNA to custom prime editing guidance RNA (pegRNA), which both stipulates the target site and encodes the anticipated editing sequence. Anzalone et al. (2019) reported that pairs of pegRNAare capable of exactly deleting 710 bps or accurately replacing a sequence of 108 bps. To date, prime editing has been applied to corn, rice, wheat, and tomato (Jiang et al., 2020; Lin et al., 2020; Lu et al., 2021a).

Genome-editing has been extensively used across plant species to study and incorporate functional mutations for crop improvement. Conversely, the integration of transgene in the genome of plants is likely to attract considerable public attention and fall under the regulatory frameworks that control use of genetically modified organisms (GMOs)(Gu et al., 2021). Conventional genome engineering methods need the transfer and combination of DNA cassettes to encode modified parts into the host genome. DNA fragments are generally degenerated but generate detrimentally effects (Kim et al., 2014). Transgene free genome editing is becoming a fast-expanding movement in biological sciences because of its advantages. This technique opened the roads to targeted genome alterations without conflict with the genome and created opportunities to deliver non-genetically modified organisms (Zhang et al., 2016a; Rather et al., 2022). However, transgene free genome editing comes across the same fundamental issues as transformation approaches. Regardless of the broader arsenal of transformation techniques, the RNA and protein delivery methodologies are less developed for plant cells than for animals. Thus, only the biolistic method and protoplast transfection could be used to generate transgene-free genome-edited plants (Tsanova et al., 2021). Protoplasts were the primary tissue successfully targeted for DNA-free genome editing through polyethylene glycol (PEG) mediated fusion. Hence, ribonucleoprotein (RNP) complex or mRNA mix with PEG and combine with the protoplast.

Trans-gene free genome editing was studied by transfecting guide RNA and Cas9 protein into protoplasts of tobacco, Arabidopsis, rice, and lettuce and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46% (Woo et al., 2015). Furthermore, to achieve DNA-free genome-edited plants, the wheat embryo has been used for particle bombardment using CRISPR/Cas9 RNAs (Zhang et al., 2016a).This study achieved highly efficient and precise DNA-free genome editing by a transient expression that produced homozygous mutant plants in the T0 generation. In addition, a recently published report by (Rather et al., 2022)revealed an efficient method of DNA-free genome editing in potato (Solanum tuberosum) protoplast using circular and linearized plasmid DNA fragments that showed high expression of the transgene and upto 95% gene editing events in protoplast derived potato calli (Rather et al., 2022).

Multiplex genome-editing technologies are versatile and powerful tools for precisely modifying numerous specific loci in the genome. In this technique, various gRNA and Cas9 enzymes are expressed at once (Wolter and Puchta, 2017), thus facilitating potent bioengineering applications and greatly improving genome-editing efficiencies (Jacobs et al., 2017). Furthermore, these approaches have significantly enhanced the likelihood of obtaining desired alterations at multiple nucleotide levels in the target genome. With numerous sgRNA targets, several genes can be modified concurrently in any crop plant. Using this technique, various traits could be introduced into new plant varieties. Furthermore, numerous individuals from many families could be targeted by combining multiple sgRNAs into a plasmid vector (Komor et al., 2017; Meng et al., 2017). The principal advantage of CRISPR is its ability to allow multiplex genome editing via editing multiple sgRNA targets in the genome. In plants, the application of multiplex genome editing has been demonstrated for improving traits like herbicide tolerance. Nevertheless, the applications are now being extended to cover multiple aspects of plant improvement including metabolic engineering and hormone biosynthesis and perception, and molecular farming capturing >100 concurrent targeting events (Armario Najera et al., 2019)Therefore, multiple genome editing technologies will accelerate creation and utilization of novel genetic variations for faster breeding of new crop varieties for unpredictable and changing climates.

The greater efficiency of CRISPR-mediated mutagenesis of crop plants allows the improvement of the high-throughput mutagenesis approach. The common use of CRISPR/Cas9 is important for designing mutant libraries to learn the genetic means behind crop improvement. In traditional mutagenesis process, a plant receives many mutations in the genomic background in addition to the targeted site. This leads to a weak association between the genotype and phenotype and further lowers the success of gene identification and cloning (Meng et al., 2017). The preparation of mutant libraries is an efficient and promising tool (Lu et al., 2017). CRISPR-Cas based mutant production has an advantage over chemical-based mutation for being more site-specific in the genome and having an strong association to the phenotype of interest. Genome-scale mutant library via CRISPR/Cas9 are now available for Arabidopsis (Peterson et al., 2016), tomato (Jacobs et al., 2017) and in rice (Meng et al., 2017). The mutant libraries are prepared by changing the 18-20 bp target binding order in the sgRNA target. Tomato transformation was carried out by transforming pooled CRISPR libraries to generate a group of mutant lines with the least transformation attempts and in less time (Jacobs et al., 2017). In this study, a single transformation attempt was performed using the CRISPR library that targeted immunity-related leucine-rich repeat subfamily XII genes resulting in inherited mutation retrieving in 15 of the 54 targeted genes.

Further, to improve productivity, they constructed a second library containing three sgRNAs per construct to target 18 genes, resulting in mutagenesis in 15 of 18 targeted genes (Jacobs et al., 2017). For the rice plant transformation, mutant libraries were generated with loss-of-function mutation (Meng et al., 2017). These plants showed phenotypic changes like lethality and sterility during their cultivation in the field. Overall, mutant libraries are a powerful tool for improving genome-editing techniques for abiotic stress management in crops. By screening mutant libraries for stress tolerance and identifying targets for genome editing, researchers can develop more effective strategies for improving stress tolerance in crops.

Overexpression of several drought-responsive genes and transcription factors increases the accumulation of signaling molecules and metabolic compounds and enhances drought tolerance in plants (Fang and Xiong, 2015; Kumar et al., 2019; Santosh Kumar et al., 2020). The expressions of drought-sensitive (S) genes enhance susceptibility in plants to drought through hormonal disproportion, declined antioxidant activities, and increasing reactive oxygen species (ROS) production. Over-expression of AREB1 has shown improved tolerance to drought stress, whereas the AREB1 knock-out mutant had higher sensitivity to drought stress (Singh and Laxmi, 2015). CRISPR/Cas9 targeted mutagenesis of SlLBD40, a lateral organ boundaries domain transcription factor that enhances drought tolerance in tomatoes compared with overexpressing transgenic and WT tomato plants; knockout of SlLBD40 by CRISPR/Cas9 enhanced the drought tolerance of tomato (Liu et al., 2020). CRISPR/Cas9-edited tomato (Solanum lycopersicum) mutant plants knock-out for SlMAPK3 gene had enhanced drought stress response (Wang et al., 2017b). Under drought, these mutant plants exhibited severe wilting symptoms, elevated levels of H₂O₂, reduced antioxidants, and increased membrane damage. These results substantiate that SlMAPK3is implicated in drought stress response in tomato plants by protecting the cell membrane. Knockout of the tomato Auxin Response Factor (SlARF4) gene improves tomato resistance to water deficit (Chen et al., 2021). Arabidopsis histone acetyltransferase 1 (AtHAT1) promotes gene expression activation by switching chromatin to a relaxed state. Improved drought stress tolerance was observed in Arabidopsis by CRISPR/dCas9 fusion with a Histone Acetyl Transferase (AtHAT) gene (Roca Paixão et al., 2019). CRISPR/Cas9-based editing of pathogenesis-related 1 (NPR1) gene in tomato exhibited drought response (Li et al., 2019) by enhancing stomatal aperture, malondialdehyde (MDA) level, H₂O₂ content and ion leakage. However, the antioxidant activity level declined more than in wild type plants. The SlNPR1 plays a significant role in directing responses against drought stress in tomato and other crop plants. Multiple SlNPR1 variants can be developed through genome editing to enhance drought tolerance in a wide range (Li et al., 2019). Drought-induced SINA protein 1 (OsDIS1), drought and salt-tolerant protein 1 (OsDST), and ring finger protein1 (OsSRFP1) genes are negative regulators of drought tolerance. Silencing these drought-responsive genes improved levels of antioxidant enzymes, decreased concentrations of H₂O₂, and increased tolerance to drought stress in rice plants (Fang and Xiong, 2015; Santosh Kumar et al., 2020). Enhanced Response 1 (ERA1) protein gene regulates plants’ ABA signaling and dehydration responses. In rice, editing of the OsERA1gene enhanced tolerance to drought stress, with the mutant plants showing increased sensitivity to ABA and stomatal closure under drought conditions (Ogata et al., 2020). OsSAPK2 is also involved in ABA-mediated stress tolerance in rice and its participation was confirmed by developing mutants using CRISPR/Cas9 with loss of function mutation. The mutants exhibited more drought sensitivity than WT plants (Lou et al., 2017).

Transcription factor OsWRKY5 inhibits the ability to withstand drought. OsWRKY5 was mostly expressed in growing leaves throughout the seedling and heading phases, and drought stress decreased its expression. Plant growth under water shortage was used as a measure of the increased drought tolerance imparted by the genome-edited loss-of-function alleles oswrky5-2 and oswrky5-3 (Lim et al., 2022). In contrast, greater susceptibility was observed under the same circumstances when OsWRKY5 was overexpressed in the activation-tagged line oswrky5-D. OsWRKY5 activity was lost, increasing sensitivity to ABA and encouraging ABA-dependent stomatal closure. OsWRKY5 genome editing increases grain production under drought stress.

The CRISPR Cas9-induced mutations in the gene encoding OPEN STOMATA 2 (AtOST2) in Arabidopsis mutants facilitated an enhanced stomatal response than WT (Osakabe et al., 2016).

Interestingly, the AtOST2 mutants had a high degree of stomatal closure (Osakabe et al., 2016). In rice, OsSRL1 and OsSRL2 gene encodes leaf tissue phenotype. The genome-modified lines having homozygous SRL1 and SRL2 mutants were found retardation in various characteristics such as the stomata number, stomatal conductance, transpiration rate, chlorophyll content, vascular bundles and other agronomic traits in comparison to wild-type one (Liao et al., 2019). Drought tolerance can be obtained through CRISPR/Cas9-based genome editing by targeting negative regulators or drought-sensitive genes. CRISPR/Cas9-based genome editing in Zea mays was carried out to enhance the expression level of the ARGOS8 gene, which negatively regulates ethylene response, for the development of drought tolerance. Such mutant plants showed improved grain yields in the field under drought-stress conditions (Hirai et al., 2007). WRKY transcription factors regulate the plant’s growth and development and involve biotic and abiotic stresses. In plants, WRKY3 and WRKY4 genes play an important role in regulating defense response to drought stress (Li et al., 2021b).

By the year 2050, more than 50% of agricultural lands may get critically salinized (Wang et al., 2003; Kaashyap et al., 2017; Jha et al., 2019). In plants, salt stress causes various physiological and morphological changes because of alterations in the expression of genes and signaling pathways (Prusty et al., 2018). The key detrimental effects of salinity stress are necrosis, untimely death of old leaves, and harsh interruption of ions in cells (Julkowska and Testerink, 2015). Several genes have been identified and characterized through CRISPR/Cas-based genome editing to improve plant salt tolerance. Knockout of AtWRKY3 and AtWRKY4 genes in A. thaliana plants using CRISPR/Cas9 exhibited significant up-regulation of genes under salt and methyl jasmonate stresses. Such double mutant plants showed sensitivity features to salinity and methyl jasmonate, such as elevation in ion leakage and reduction in antioxidant activities, including peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD)(Chen et al., 2021) ( Table 1 ). Also, the importance of the Acquired Osmotolerance (AtACQOS) gene provided tolerance against salt stress in Arabidopsis is characterized by CRISPR-generated mutants (Kim et al., 2021). CRISPR/Cas9-based knock-out mutants of abscisic acid (ABA)-induced transcription repressors (AITRs) genes conferred salt stress tolerance in soybean (Glycine max) plant (Wang et al., 2021b).These mutant plants showed increased ABA sensitivity and produced longer roots and shoots than WT plants. Similarly, knock-out mutants of GmDrb2a and GmDrb2b genes showed enhanced salinity stress tolerance in G. max (Curtin et al., 2018). CRISPR/Cas9-mediated editing of OsRAV2 gene expression was induced by the regulatory function of the GT-1 element in rice and showed tolerance to salt stress (Duan et al., 2016). CRISPR mutants with loss of function of SnRK2 and osmotic stress/ABA-activated protein kinases SAPK-1 andSAPK-2 genes showed resistance to salt stress in rice (Lou et al., 2017). In several other studies, the development of CRISPR-mutants in rice to develop salt stress tolerance plants through knock-out of OsDST (Santosh Kumar et al., 2020), OsNAC45 (Zhang et al., 2020), AGO2 (ARGONAUTE2) (Yin et al., 2020), Rice type-B response regulator (OsRR22) (Zhang et al., 2019), and OsBBS1 (bilateral blade senescenc1) (Zeng et al., 2018) have been carried out. Knock-out mutants of the TaHAG1 gene of wheat plants generated through CRISPR/Cas9 showed enhanced salt tolerance (Zheng et al., 2021). Alam et al. (2022) generated the gene edited rice mutant plants through targeting OsbHLH024 gene to study its role under salt stress condition. The deletion of one nucleotide base was observed in the osbhlh024 mutants its exposure under salt stress resulted in significantly enhance in total chlorophyll content and shoot biomass (Alam et al., 2022).

Violaxanthin de-epoxidase (VDE) plays a critical role in plants’ ABA biosynthesis, growth, and stress responses. In rice, the functional benefits of OsVDE in salt tolerance are validated. Gene-editing targeting OsVDE loci in overexpressed transgenic rice was found to have a higher ABA level, stomatal closure percentage and survival rate than the wild type under seedling stage salt stress. (Wang et al., 2003). Several plant transcription factor family genes are involved in the salt stress response. NAC transcription factor coding gene, OsNAC041, confirmed its importance for germinating seeds under salt stress. Osnac041 mutant obtained by CRISPR/Cas9 method showed increased salt sensitivity compared to the wild plants (Bo et al., 2019). Transcription factor OsDOF15 positively regulates primary root elongation by regulating cell proliferation in the root meristem via restricting ethylene biosynthesis. Loss-of-function of OsDOF15 impaired primary root elongation and cell proliferation in the root meristem (Qin et al., 2019). Some instances where editing on the negative regulator for salt stress tolerance are OsPQT3 and the DELLA protein SLENDER RICE1 (SLR1). In rice, OsPQT3 knockout mutants displayed enhanced oxidative and salt stress resistance to elevated expression of OsGPX1, OsAPX1 and OsSOD1 under salt stress (Alfatih et al., 2020), and the loss of function of SLR1 promoted mesocotyl and root growth, specifically in the dark and under salt stress (Mo et al., 2020).

High temperature or heat stress is one of the major abiotic stresses that become a severe problem in agricultural production in several regions of the world that causes global warming (Tubiello et al., 2007). Plants react to heat stress by activating complex molecular networks, including heat stress-responsive gene expression, signal transduction, and metabolite production (Jha et al., 2017a). With the advancements in functional and structural genomics techniques in plants, various heat stress-associated genes have been identified and characterized to enhance heat tolerance with advanced biotechnological tools. The Heat shock proteins (HSPs) and heat shock transcription factors (HSFs) are crucial gears and function through the heat stress-response signal transduction pathway, which is linked to ROS accumulation (Awasthi et al., 2015). Hence, heat stress tolerance can be improved by enhancing the ability of plants to detoxify ROS components (Parmar et al., 2017). This indicated that enhanced tolerance could improve crop plants’ antioxidant activities. Heat-induced gene expression and metabolite biosynthesis significantly enhanced heat tolerance in plants. Among all the genome-editing approaches, CRISPR/Cas9 is a revolutionary technique for genome editing in a precise manner to learn the molecular pathways associated with heat stress and improve crop heat tolerance (Duan et al., 2016). Tomato (Solanum lycopersicum) is considered an ultimate model to test CRISPR/Cas9-based genome editing because it can endure competent transformation to grain quality enhancement (Pan et al., 2016). Currently, CRISPR/Cas9-based genome editing of the heat-sensitive gene, SlAGAMOUS-LIKE 6 (SIAGL6), in tomatoes was generated for heat tolerance, enhancing fruit setting under heat stress conditions (Klap et al., 2017).

In tomatoes, the SlMAPK3 gene belongs to the mitogen-activated protein kinase family and participates in response to diverse environmental stress functions. CRISPR/Cas9-mediated genome editing resulted in slmapk3 mutants showing enhanced thermo-tolerance compared to WT plants and implying its role as a negative regulator of thermo-tolerance (Yu et al., 2019). BRZ1 positively regulates ROS production in the apoplastic region in tomatoes and serves as a component for heat tolerance. This has been validated from the CRISPR/Cas9-based bzr1 mutants that showed impaired H₂O₂ production in apoplast and heat tolerance by declined Respiratory Burst Oxidase Homolog 1(RBOH1)(Yin et al., 2018). Development of CRISPR/Cas-mediated HSA1 (heat-stress sensitive albino 1) mutants of tomato showed increased sensitivity to heat stress compared to wild-type plants (Qiu et al., 2018). In maize, CRISPR mutants of the thermosensitive genic malesterile 5 (TMS5) gene improved thermosensitive male-sterile plants (Li et al., 2017). In lettuce, the germination of the seeds at a higher temperature was achieved through knockouts of NCED4, a key regulatory enzyme in the biosynthesis of ABA. Therefore, mutants of LsNCED4 could be commercially valuable in production areas with high temperatures (Bertier et al., 2018).

Low temperature is a key abiotic stressor that adversely influences plant growth and productivity (Jha et al., 2017b). In plants, cold stress tolerance is a highly intricate trait concerning several diverse cell compartments and metabolic pathways (Hannah et al., 2006). Conventional breeding approaches have achieved adequate success in enhancing the cold tolerance of significant crop plants relating to inter-specific or inter-generic hybridization. Cold stress causes damaged seedlings, poor growth, and a low germination rate in rice. It can also decrease grain yield at reproductive phage in rice (Koseki et al., 2010; Shakiba et al., 2017). CRISPR/Cas9 is an attractive and accessible technology for developing non-transgenic genome-edited crop plants to overcome climate change and ensure future food security (Mo et al., 2020). In rice, editing is guided to knockout some negative regulator transcription factors to increase plant tolerance for cold. OsMYB30 is a transcription factor that binds to the promoter of the β-amylase gene and negatively influences cold tolerance. Under cold stress, OsMYB30 makes a complex with OsJAZ9 and inhibits the expression of β-amylase gene, thus affecting starch degradation and maltose accumulation, which may contribute to increasing cold sensitivity (Lv et al., 2017). CRISPR/Cas9-based genome editing of three genes, OsPIN5b, GS3, and OsMYB30, mutated simultaneously and showed enhanced yield and tolerance to cold stress (Zeng et al., 2020). Plant annexins are involved in the regulation of plant development and protection from environmental stresses: Rice annexin genes OsAnn3 and OsAnn5 are positive regulators of cold stress tolerance at the seedling stage. The Knocking out of OsAnn3 and OsAnn5 resulted in sensitivity to cold treatments (Shen et al., 2017). In addition, OsPRP1 enhances cold tolerance in rice by modulating antioxidants and maintaining cross talk through signaling pathways. Knockout of OsPRP1 induced cold sensitivity in rice, and mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, ABA, and ascorbic acid (AsA) content relative to WT under low-temperature. Tomato plants are sensitive to chilling stress; therefore, their fruits are more prone to be damaged by cold stress. CRISPR/Cas9-based cbf1 mutants showed that C-repeat binding factor 1 (CBF1) protects the tomato plant against chilling/cold damage and decreases electrolyte leakage (Li et al., 2018b). These plants also showed a higher accumulation of hydrogen peroxide and indole acetic acid, thus, providing tolerance to cold stress in tomato plants. The expression of ten transcription factors from the WRKY family was observed to be two-fold higher under cold stress (Chen et al., 2015). In Cucumber, over-expression of the CsWRKY6 gene showed enhanced tolerance to cold stress and sensitivity to ABA and proline accumulation (Zhang et al., 2016b). RNA sequencing of Brassica napus revealed various genes from the WRKY family that play an important role in cold response (Ke et al., 2020). The SlNPR1 expression and protein content was found to be increased under low temperature (4°C) in tomato. CRISPR/Cas9-mediated genome editing of SlNPR1 induced the symptoms of chilling injury in tomato plant that was substantiated by the accumulation of hydrogen peroxide (H₂O₂), superoxide anion (O2−), and malonic dialdehyde (MDA), and reduction in proline content, antioxidant enzymatic activities, and soluble protein content (Shu et al., 2023).In strawberry, overexpression of FvICE1 gene resulted in tolerance to cold and drought at the phenotypic and physiological levels. However, CRISPR/cas9-mediated gene edited strawberries mutant showed lower cold and drought tolerance. These results suggested that FvICE1 functioned as a positively regulator of cold and drought (Han et al., 2023).

Heavy metal stress is one of the key problems that adversely affect the agricultural productivity of plants (Jha and Bohra, 2016). Plants practice oxidative stress upon contact with heavy metals, leading to cellular injury (Yadav, 2010). Additionally, the accumulation of metal ions in plants perturbs cellular ionic homeostasis. Therefore, plants have developed detoxification mechanisms to reduce heavy metal exposure’s damaging effects and accumulation. Such mechanisms involve controlled elimination of toxic ions from roots, metal uptake, efficient neutralization of metal ions in the protoplast, and appropriation or translocation to remote organs (Sruthi et al., 2017). Various genes direct these mechanisms to enhance tolerance to heavy metal stress (Hasanuzzaman et al., 2019). For example, the loss-of-function mutant of γ-glutamylcyclotransferase showed defensive characteristics against heavy metal toxicity suggesting that the loss-of-function mutants of OXP1 and γ-glutamylcyclotransferase demonstrate heavy metal and xenobiotic detoxification due to increased glutathione (GSH) accumulation (Paulose et al., 2013). Therefore, developing CRISPR/Cas9-mediated mutants would be useful to fight against the heavy metal stress in plants. Recently, Baeg et al. (Baeg et al., 2021) developed oxp1/CRISPR mutant Arabidopsis plants that showed resistance to Cadmium, suggesting an improved capability of heavy metal detoxification in mutant plants compared to WT Col0 plants. Consequently, this study showed a way to confer resistance to xenobiotics and heavy metals in plants by indel mutations using the gene-editing method (Baeg et al., 2021).

In rice, the roots absorb Cadmium from the soil with the transporters OsNramp1, OsNramp5, and OsCd1. OsHMA3 plays the role of Cadmium sequestration into root vacuole and negatively regulates xylem loading, and OsLCT1 is involved in Cadmium transport to the grains (Chen et al., 2019a). Manipulating the expression of these transporter genes by genome editing has found some success in reducing Cadmium in the grain crop. The CRISPR/Cas9-based mutants of OsNramp5 and OsLCT1 genes resulted in allow Cadmium level in rice (Lu et al., 2017; Tang et al., 2017a; Wang et al., 2017b). Similarly, OsARM1 regulates As-associated transporter genes in rice. It is expressed in the phloem of the vascular bundle in the basal and upper nodes. Knock-out of the OSARM1 by CRISPR improves tolerance, while its overexpression has increased sensitivity to As (Baeg et al., 2021). Cs+-permeable OsHAK1 transporter in rice is the major pathway for Cs+ uptake and translocation. To minimize the radioactive caesium (Cs) uptake by rice plants in Fukusima soil contaminated with 137 Cs+, the CRISPR-Cas system was used to obtain transgenic plants lacking OsHAK1 function. TheOsHAK1knock-out plants displayed strikingly reduced levels of 137 Cs+ in roots and reduced radioactive caesium contents (Nieves-Cordones et al., 2017). Another instance of using the CRISPR-Cas-based editing in rice is to know the function of a potential target, OsPRX2, for improved potassium deficiency tolerance. OsPRX2 is known to reduce the production of ROS in a K+ limiting condition. It was found overexpression of OsPRX2 causes the stomatal closing and K+ deficiency tolerance to increase. At the same time, knockout of OsPRX2 leads to serious defects in leaves phenotype and the stomatal opening under the K+-deficiency tolerance (Mao et al., 2019).

Weeds are global agricultural constraints that threaten crop production by posing stiff competition for the main crop for nutrients, soil moisture, light, space, and CO2. Weed growth is one of the key factors that influence the quality and yield of crop plants (Sundström et al., 2014). Several approaches have been tried to eradicate weeds (Green, 2014). The herbicide application is the key tool used for weed management in recent crop production systems (Gage et al., 2019). Herbicide tolerance is one of the most important traits of crop plants that advance farming techniques and the productivity of crop plants. CRISPR/Cas9-based genome editing to develop herbicide-resistant crop plants is now the ideal system to control weeds (Toda and Okamoto, 2020). Herbicide-tolerant crop plants showed higher yields and could minimize toxicity to the environment and our body three times compared to crops cultivated through the conventional method(Dong et al., 2021). This should be adapted as an important practice for high-scale farming; cost-effective and requires less effort to develop transgene -free wheat germplasms containing herbicide tolerance mutations that provide tolerance to aryloxyphenoxy propionate-, sulfonylurea-, and imidazolinone-type herbicides by base editing the acetyl-coenzyme A carboxylase and acetolactate synthase (ALS) genes (Zhang et al., 2020). Acetolactate synthase 1 (ALS1) is a crop plant’s most important enzyme for herbicide tolerance. CRISPR-mediated genome editing technique has also been applied to introduce herbicide tolerance in crop plants [ Table 3 ]. Some examples of crops that have been edited for ALS gene-based herbicide resistance include tomato, soybean, rice, wheat, maize, watermelon, oilseed rape, tabaco, potato and arabidopsis (Dong et al., 2021). Overall, genome editing offers a powerful tool for developing herbicide-resistant crops by modifying key genes such as ALS. A new herbicide tolerance trait has been incorporated in oryza sativa through CRISPR-based editing of the OsALS1 gene (Kuang et al., 2020). Mutants of rice generated by developing a new allele (G628W) by G-to-T transversion at 1882 position in the OsALS gene showed strong herbicide tolerance. The progenies of rice mutants were transgene-free and harbouring homozygous alleles (G628W) that were agronomically similar to the wilting type. These mutant plants of rice conferred resistance to imazethapyr (IMT) and imazapic (IMP) herbicides (Wang et al., 2021a). Lu et al. (2021) investigated that in rice, new genes and traits can be developed through designing large scale genomic duplication or inversion by CRISPR/Cas9 mediated genome editing. They showed that high transcript accumulation of CP12 and Ubiquitin2 genes were found in leaves and the expression level of HPPD and PPO1 was upregulated by 10 folds in edited plants with homozygous structural variations alleles and resulted in herbicide resistance in field trials without hampering the yield and other agronomically important traits (Lu et al., 2021b). CRISPR/Cas9-mediated genome editing can be useful to generate herbicide-tolerant crop plants. The CRISPR/Cas9-based targeted mutagenesis of three genes ALS (acetolactate synthase), EPSPS (5-Enolpyruvylshikimate-3-phosphate synthase), and pds (phytoene desaturase) conferred herbicide resistance in Solanum lycopersicum cv. Micro-Tom (Yang et al., 2022). These herbicide tolerance traits offer a potentially powerful approach to weed management. Thus, the CRISPR-based genome-editing tool could precisely advance the engineering of herbicide-resistant genes in crop plants. However, it is important that the use of such crops must be carefully monitored to prevent the development of herbicide-resistant weeds and other unintended consequences.

Genome-editing technology has been considered a significant tool and revolutionized the development of abiotic stress-tolerant crop plants in recent decades. Genome-editing approaches could demonstrate plant tolerance to abiotic stresses by targeting stress-responsive genes, sensitive genes or negatively regulating genes and activating positively regulated genes that control abiotic stress responses. Known positive and negative regulators of a certain trait are ideal targets for crop improvement. Knock out in structural genes, regulated genes, transcription factors and promoter regions produce altered and broken protein products and generate stress tolerant phenotype through modification of biochemical pathways, metabolite profile and physiology. For example, mutagenesis of a structural gene semi-rolled leaf1, 2 in maize confers curled leaf phenotype and drought tolerance by influencing protein expression patterns and ROS scavenging in rice (Liao et al., 2019). In tomatoes, SlMAPK3 acted as a negative regulator of defense response to heat stress, and the knockout of SlMAPK3 enhances tolerance to heat stress involving ROS homeostasis (Yu et al., 2019).

Similarly, knock out of the negative regulator of drought tolerance SlLBD40, a lateral organ boundaries domain transcription factor, enhances drought tolerance in tomatoes (Liu et al., 2020). Figure 3 demonstrated these negative regulators of the stress-responsive genes and their inhibitors by editing them to improve stress tolerance for salinity, drought, cold, etc. In addition to the knockout and allele insertion-deletion, the CRISPR editing tool also generates tolerant plants by modulating the genomic aspects through transcript activation, epigenetic modification by chromatin unwinding, and swapping promoter regions with HDR (Li X et al., 2022). These Arabidopsis mutant plants were epigenetically modified for ABA/AREB1 responsive transcription factor by chromatin unwinding, contributing to improving drought tolerance (Roca Paixão et al., 2019). Similarly, the CRISPR/Cas9 transcript activation of Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1) leads to increased leaf areas and enhanced tolerance to drought stress (Park et al., 2017). CRISPR/Cas9 system has also witnessed the improvement of drought tolerance by creating novel allelic variation in maize using HR-mediated promoter switching (Shi et al., 2017). Maize ARGOS8 is a negative regulator of ethylene responses. ARGOS8 variants created by replacing its promoter with GOS2 increased plant yield under drought (Shi et al., 2017). In Figure 3 , we demonstrated the probable mechanisms of CRISPR/Cas9-based editing of sensitive genes and negative regulators by which plants perceive environmental stresses, transduce their signals and, throughout a sophisticated and finely coordinated response, integrate stress signals, hormonal metabolism and adaptive responses that lead to tolerance. Various structural and regulatory genes (e.g., non-coding RNAs) are known for responding to diverse environmental stresses that can be targeted using CRISPR/Cas9 to improve stress tolerance in agronomically important crop plants (Zafar et al., 2020). Besides developing knockout mutants of individual genes through the CRISPR/Cas system, it can also be used to activate the expression of important candidate genes involved in abiotic stress tolerance.

Additionally, the expression of sensitive genes enhances abiotic stress responses in plants through impaired biochemical (chlorophyll content, changes in antioxidants activities, increased ROS production, ion leakage, lipid peroxidation), physiological (reduced biomass, photosynthetic rate, and higher transpiration rates) and phenotypic (flowers/pods abortion) responses that result in reduced crop yield. Remarkably, CRISPR/Cas9-mediated genome editing approach offers better stress resilience in crop plants through silencing or modification of target genes controlling biochemical, physiological, and morphologicalcomponents. Furthermore, these genetically edited CRISPR plants show elevated photosynthetic capacity, increased root length and density, increased biomass, increased nutrient accessibility, stomatal closure, higher chlorophyll content, reduced transpiration rate, the structural adaptation of membranes, and increased relative water content, decreased electrolytic leakage and malondialdehyde content, the reduced metal accumulation that results in abiotic stresses tolerance in plants Figure 3 .