1 Introduction

With the continuous rise of the world population (60%), there will be a scarcity of food, and climate change has adversely affected it (Zafar et al. 2020), and that has put more pressure on the agriculture sector (Suweisa et al. 2015). Besides abiotic stresses (salinity, drought, toxicity of heavy metals, and hot temperatures) and climate change, humans (harmful activities) have also had adverse effects on cultivated regions (Zafar et al. 2020). Both types of stresses (biotic and abiotic) have minimized (70%) food production (Ahmad et al. 2020). Therefore, more advanced and resistant crops should be developed against such stresses to overcome food shortages (Driedonks et al. 2016) and enhanced up to 70–85% as an estimated increase of 9.7 billion could occur in the population by 2050 (Zaidi et al. 2016). Traditional actions take more time to develop resistant crops (Adeyinka et al. 2023). So, it is needed to adopt such genetic tools (i.e., genome editing) for crop improvement and to overcome stresses (Driedonks et al. 2016). Besides accepting GM crops (genetically modified, stress-resistant and nutritive) in certain pockets of the world (Shukla et al. 2018), it has developed several crops known as golden rice and Bt-cotton that represent the gene revolution (Napier et al. 2019).

Several tools and the three main divisions of CRISPR (meganucleases, zinc finger nucleases (ZFNs), transcription activator like nucleases (TALENs), CRISPR-Cas9, and clustered regularly interspaced short palindromic repeats) have been developed for gene editing (deletion, insertion, or changing a particular pattern of DNA), especially in plants (Kumar et al. 2023) or the 4OMT2 gene knocking out in Papaver somniferum (regulating the biosynthetic pathway of benzylisoquinoline alkaloids) (Alagoz et al. 2016). Among them, CRISPR-Cas9 (productive, quickest, and most precise) has wide usage (acquired from the natural resistance system found in bacteria against virus attacks), which identifies and cleaves the corresponding DNA pattern along with CAS enzymes (Chen et al. 2019a, b). This technique has direct or indirect influence over quantitative genes to withstand abiotic shocks (Mushtaq et al. 2018; Hossain et al. 2022). The approach of foreign DNA will enhance social acceptance rates. Commercially, tomatoes with more γ-aminobutyric acid than typical ones are available in Japan (edited by CRISPR-Cas9) (Waltz 2022). This technology enhanced the activities of stress tolerant genes and increased production (Zafar et al. 2020).

In this review, we summarize most potential applications of the CRISPR/Cas9-mediated genome editing approach in crop plants aimed at managing abiotic stresses such as drought, salinity, cold, heavy metal, and heat resistance in sustainable agriculture, and discuss the advantages, and limitations.

2 Background History of CRISPR/Cas9

Prokaryotes genomes (bacterial and archaeal) have 40–90% CRISPR to survive against attackers through defence mechanisms (Gajardo et al. 2023). In mid-1980, editing of genes was initiated, and several crops (wheat, rice, cotton, maize, potato, tomato, and vice versa) were developed (Nascimento et al. 2023). Beside the extended period taken by zinc finger nucleases (ZFNs) and termed TAL effector nucleases (TALENs) to target DNA binding proteins (Ceasar et al. 2016), TALENs, ZFNs, and CRISPR-Cas9 are very fruitful against numerous stresses (Nongpiur et al. 2016). Addition and removal of a particular pattern happen by the NHEJ (non-homologous end-joining) repair pathway (Fig. 1), where double strand breaks (DSBs) form from cleavage by order-particular nucleases after pattern detection and are remoulded by non-homologous end joining (NHEJ) and HR (homologous recombination) (Belhaj et al. 2015). Restrictions of DNA binding proteins (ZFNs and TALENs) were identified due to CRISPR’s beginning (Kumar et al. 2023). There are two components of the CRISPR-Cas9 structure (namely CRISPR RNAs and Cas9 proteins). GM crop production and plant function could be described by CRISPR/Cas9 (Tripathi et al. 2020).

Fig. 1
figure 1

Procedure of CRISPR-Cas9 (driven by RNA) producing DSB in the required pattern of DNA and its suitable renovation paths (HDR and NHEJ). Double strand breaks (DSBs), protospacer adjacent motif (PAM), non-homologous end joining (NHEJ), homology-directed repair (HDR), non-homologous double-stranded oligodeoxynucleotide (dsODN)

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3 CRISPR-Cas9 Tool: A RNA-Directed Nuclease for Genes Editing

Functions and the beginning of details about CRISPR/Cas9’s modular nature for genome editing have been provided in several reviews (Zhong et al. 2024). Target DNA (through Watson–Crick base pairing) could be detected by Cas9 nuclease-mediated cleavage (sgRNA) (Zhong et al. 2024), and its pattern (5′-N) could be altered through Cas9 nuclease. sgRNAs (20–22 nucleotides; small size) are oligonucleotides, which represents their high effectiveness without any issues, and the presence of protospacer adjacent motif (PAM), NGG, or NAG site immediately 3′ is necessary for a targeted spot. So, CRISPR-Cas9 has a highly advanced role in plant biology due to its efficiency in targeting various genes.

4 Components of CRISPR Gene Editing System

Small guide RNA (sgRNA) and Cas9 nuclease are the two key constituents of the CRISPR-Cas system represented in Fig. 2.

Fig. 2
figure 2

DNA sequence. The gRNA Target (grey box) next to it there is the PAM (GGG, N can be A/T /G orC) and then the single guide RNA (sgRNA) as a blue arrow. Protospacer adjacent motif (PAM)

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4.1 sgRNA (Guide RNA)

It consists of two RNA parts along the Cas9 genes known as small CRISPR RNA (crRNA) (orders non-coding RNA constituents) and trans-activating CRISPR RNA (tracrRNA). Targeting pattern and cleaved position are detected by crRNA, while their arrangements are supported by tracrRNA (artificially bonded), and sgRNA has more efficiency than the other two due to further advancements. While minimizing off target influences, truRNA (transcribed guide RNAs) has a great role in Cas9 upgrading (Fu et al. 2014). A synthetic gene (Ribozyme RGR) develops the required gRNA through self-catalyzing cleavage (RNA molecules formed by ribozyme patterns) (Gao and Zhao 2014). As this synthetic gene is easily defined by the promoter, it provides quick details about sudden changes and gene editing, while various targets could be achieved by polycistronic tRNA-gRNA (composed of tRNA-gRNA with various spacer patterns) (Xie et al. 2015).

4.2 Cas9 Nuclease

sgRNA has two smaller RNAs (crRNA and tracrRNA) bent together to efficiently target outsider DNA through Cas9 (Li et al. 2024). Through two pathways, non-homologous end joining (NHEJ) and homology-directed repair (HDR), CRISPR-Cas9 shows immunity in cells (Gaj et al. 2013) and is helpful in gene alteration at a particular point. DSB fusion occurs through NHEJ (either removing or adding nucleotides) (Cong et al. 2013). Undeveloped proteins denature (not workable) as a result of frame shift changes (if editing of nucleotides is not divisible by 3). DSBs (double stranded breaks) are formed by the Cas9 protein (cut DNA), which further forms two sections (α-helical identifying lobe known as REC and a NUC called the nuclease lobe) with target DNA and sgRNA along the CTD (C-terminal domain), HNH, and RuvC spheres. There are 3-α helix domain (Hel-I, Hel-II, and Hel-III) in the REC section. Cas-9 particular binds (which consist of PAM points) are defined by CTD. HNH nuclease has ββα-metal fold carrying a 1-metal ion cleavage methodology, while RNase H and HNH nuclease are alike and possess 2-metal ion catalytic procedures. Thus, the innate Cas9/sgRNA approach is used in crops for gene editing and different gene identification techniques (Li et al. 2018a, b).

5 Concepts and Mechanisms of CRISPR-Cas9 Genome Editing Technology

Islam (2019) describes all aspects of CRISPR. Gene patterning in Escherichia coli (alkaline phosphatase isozyme) causing amino peptidase transformation was done by Japanese scientists (in the late 1980s), who observed an irregular downward gene replication pattern. The CRISPR name was highly accepted by researchers due to its abbreviation, which openly defines recurrence features (Jansen et al. 2002), and the association of CRISPR was shown with conserved CRISPR (CRISPR-associated genes; Cas), while its absence was observed in species without CRISPR units. These genes (cas1–cas4) were found scattered in CRISPR loci, showing CRISPR’s beginning (Jansen et al. 2002). Similarly, relative gene inquiry (archaea and bacteria) also noted these genes (encoding nucleases and helicases) presence, which represents their role in DNA metabolism (Makarova et al. 2002). Such proteins, known as RAMPs (Makarova et al. 2002), have repairing abilities connected with CRISPR arrangement (Haft et al. 2005).

Due to compatibility between host and foreign DNA, and also due to RNAi (eukaryotic RNA interference), CRISPR can split DNA and survive against contagion (through the RNA guide method) (Makarova et al. 2006). Cas9 nuclease work under the type I CRISPR locus (Escherichia coli) shifts to minute crRNAs with sole parts (Brouns et al. 2008). DNA is directed by Cas nuclease in Staphylococcus epidermidis rather than RNA, as CRISPR restricts plasmid conjugation (Marraffini and Sontheimer 2008). PAM (pattern motif at CRISPR end) (Mojica et al. 2009) directed Cas9-facilitated inference (Deveau et al. 2008). In Pyrococcus furiosus, target RNA has a role in splitting (via type III-B CRISPR-Cas inquiry) (Hale et al. 2009, 2012). Actual details about CRISPR were found in 2010. The CRISPR-Cas9 system in S. thermophilus causes DNA double-strand breaks (DSBs) on the PAM (3-bp location of DNA), which results in the appearance of new microbial strains (Garneau et al. 2010).

Streptococcus pyogenes extra-minute RNA (tracrRNA) was also seen to create settled crRNA (through CRISPR interference) (Deltcheva et al. 2011). CRISPR-Cas loci (S. thermophilus) were inserted in E. coli to successfully create an immune response (Sapranauskas et al. 2011). During 2012, CRISPR was described as being copied into RNA following splitting into Cas protein-led dsDNA due to RNA (Gasiunas et al. 2012). CrRNA binding along with tracrRNA led to sgRNA formation and DNA cleavage (Gasiunas et al. 2012). Strategies of CRISPR-Cas9 have been efficiently utilized in many valuable agronomical crop plants (Fig. 3).

Fig. 3
figure 3

Strategies of CRISPR-Cas9 have been efficiently utilized in many valuable agronomical crop plants

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Three stages of CRISPR-Cas9 (type II) methodology observed in bacteria towards protection have been displayed in Fig. 4.

Fig. 4
figure 4

The CRISPR-associated (Cas) system in bacteria plays a natural role in the clustered regularly interspaced short palindromic repeats (CRISPR) system. Protospacer adjacent motif (PAM), small CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA)

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5.1 Adaptation or Spacer Acquisition

Adaptation is composed of protospacer assortment and spacer assimilation (analyzing new recurrence). Nowrousian et al. (2010). The needed pattern is 5′-NGG-3′ (kinds I and II methods).

5.2 Handling crRNA and Maturation or Biogenesis

Biogenesis (pre-crRNA processing with various Cas proteins) of transcript tracrRNA led to mature crRNA from premature forms and crRNA-tracrRNA duplex making (Ma et al. 2016). CRISPR-associated complexes are formed by Cas proteins fusing crRNA to attach to the required DNA.

5.3 Interference

Immunity against outside outbreaks became active when cRNA led the Cas enzyme complex to detect a corresponding pattern. So, inhibition of self-targeting CRISPR occurs with PAM pattern detection (via the crRNA-tracrRNA duplex). As a result of the duplex, Cas9 nuclease fused and split, leading to DSB formation. There should be proper pattern balance (between target and spacer) along the PAM pattern. The Cas9/sgRNA complex, after recognition, attaches to target DNA (resembling sgRNA). DSB (due to 3 bp upward PAM) form which gets recovered by host restoration methodology, otherwise useless genes due to NHEJ and HDR.

6 CRISPR-Cas Based Gene Mutagenesis

Several hardens, like trait complexity and useful gene categorization, have limited the development of crops against abiotic stresses (Scheben et al. 2017), such as < 1% useful gene identification in maize (Andorf et al. 2015). Certain genes responsible for indicating, transcription elements, membrane shielding, and secondary metabolites have been studied, while numerous traits (such as post translational and transcriptional elements, protein-DNA, and protein–protein connections) are not described, which are needed to understand while developing crops. Two methods, random T-DNA mutations and confined cut of genes pointing could be used to adjust abiotic stress-resistance genes (Hussain et al. 2018), but they require more time and labor. Using PAM and sgRNA (target whole genome), CRISPR is working to edit gene patterns (single or multiple) to produce mutations (Sander and Joung 2014), while sgRNA detects plant appearances (via cloning and sequencing) against abiotic stress (used as a binary vector) (Sedeek et al. 2019). Various CRISPR-Cas9 benefits (pointed and biallelic changes, fast gene finding, and gene multiplexing) have been observed over other mutations (chemical and T-DNA). Abiotic stress resistance using various variants (Cas9 and Cas12a) could be created (Zetsche et al. 2015), especially in transgene-free edited plants using CRISPR/Cas9. CRISPR/Cas9 (after mutation) could be removed through segregation. dCas9 or Cas3 could be used as a result of pleiotropic effects (constant change), and such effects will be minimized while using Cas13a and Cas13b (RNA editing). Climate hardy crops (such as rice) could be developed through CRISPR/Cas (Lu and Zhu 2017).

7 Role of CRISPR in Crop Improvement and Growth

Yield declined (42%), especially due to biotic stress (15% global food decline) rather than other factors (Islam et al. 2016). 24 crops have been modified using CRISPR-Cas tools to withstand stresses (Ricroch et al. 2017) and enhance yield (Bhowmik et al. 2019). Numerous gene modifications at once are done by CRISPR-Cas technology (Molla et al. 2020), with various methods to use in crops (Islam et al. 2020), while also solely developing species. Several crops that have developed resistance against abiotic stress through this technique are presented in Fig. 5.

Fig. 5
figure 5

Studying multiple aspects through CRISPR-Cas9 to develop stress tolerant crops

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Three genes (brassinosteroid insensitive 1, jasmonate-zim-domain protein 1, and gibberellic acid insensitive) were modified by Feng et al. (2013), while Mao et al. (2013) modified magnesium-chelates subunit I (albinism-related). Similarly, such genes were modified at various points (multiplex CRISPR-Cas9) (Lowder et al. 2017). PDS3, AtFLS2, CYCD3, RACK1, and several other genes were also edited (Li et al. 2013). Changes could be different due to various factors (fusing power of sgRNA and chromatic assembly). Germline-causing changes were identified through several methods of floral dip mode of transformation (using tissue-definite agents and terminators) (Wang et al. 2015), such as 50 regulatory patterns worked in such genes (SPOROCYTELESS, DD45, and LAT52) development (Mao et al. 2016). Under high temperatures, a 5 bend (body tissues) and 100 bend (germ tissues) editing rate has been observed (LeBlanc et al. 2018). Production in rice has been improved to make it resistant against various stresses (Islam 2019) by modifying genes (phytoene desaturase (9%), betain aldehyde dehydrogenase, and OsMPK5), which was considered a great achievement (Jagodzik et al. 2018). The efficiency of the CRISPS tool was recognized by DSB production in crop cells through sgRNA practice (Miao et al. 2013). Oxygenase and LAZY genes have also been edited.

 Polyploidy plants represent efficient multiplex gene modification (Wang et al. 2018), which in wheat (ethylene responsive element 3 and dehydration responsive element fusing protein 2) (Kim et al. 2018), led to unique offspring development. Hexaploid wheat also represents gene editing (via appropriate practice) (Bhowmik et al. 2018). Phytoene desaturase gene knockout was done efficiently (40.7%–52.0%) in watermelon (Tian et al. 2017a, b). Furthermore, the ALS gene was modified to withstand herbicide toxicity. GmFT2 (the flowering time-responsible gene) was edited, leading to late flowering irrespective of day length (Cai et al. 2018). GmFEI2 and GmSHR were edited for hairy root system improvement (Cai et al. 2015).

CRISPR-Cas9 is playing a transformative role in enhancing the nutritional value of crops. This application is particularly important given the global nutrition challenges, such as micronutrient deficiencies. CRISPR-Cas9 can be utilized to increase the levels of essential vitamins, minerals, and other beneficial compounds in crops, thereby improving their nutritional profile (Molla et al. 2020). One notable example is biofortification, where crops are genetically engineered to accumulate higher levels of micronutrients (Bhowmik et al. 2019). For instance, CRISPR-Cas9 has been used to increase the iron content in rice, addressing iron deficiency, which is a major global health issue. Similarly, the technology can be employed to enhance the levels of vitamins, such as vitamin A, in crops like rice (Golden Rice) and banana, which is crucial for preventing vitamin A deficiency related diseases. In addition to increasing micronutrients, CRISPR-Cas9 can also be used to modify the composition of macronutrients to improve the overall nutritional quality of crops (Caiet al. 2015). This includes increasing the content of essential amino acids, unsaturated fatty acids, and dietary fibers, as well as reducing anti-nutritional factors or allergens. For example, using CRISPR-Cas9 to reduce gluten in wheat can be beneficial for individuals with gluten intolerance or celiac disease (Jouanin et al. 2020).

8 Uses of CRISPR/Cas9 for Advancing Crop Genomics

Due to the simplicity of CRISPR-Cas9 (accurate) and its effectiveness, it has been widely used in genetic editing and breeding of crops (exclusive cultivars and successions), rather than traditional breeding procedures. According to Bortesi and Fischer (2015), it plays a significant role in pyramid breeding that employs simultaneous multiplex editing, such as gene elimination by NHEJ.. In addition to CRISPR, other technologies (epigenetic modification and gene expression regulation) play a role in gene editing in agriculture to improve production and resistance to pest infestations.. Irrespective of rules regarding genetically modified organisms, CRISPR/Cas9 inserts genes (by agro-infiltration, viral infection, or preassembled Cas9 protein-sgRNA ribonucleoprotein transformation) in crops without transgenic impression (Woo et al. 2015). Figure 6 shows the CRISPR-Cas9 mechanism for editing crop genes.

Fig. 6
figure 6

Diagram of a typical knockout editing experiment in a crop plant, with associated timeline

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9 Gene Editing Through CRISPR/Cas9 Influences Production and Plant Survival Against Abiotic Stresses

Crop yield declines as a result of numerous abiotic stresses. Table 1 represents the number of genes activated during abiotic stress along with plant alteration approaches to cope with abiotic stresses. More than 20 crops have been modified against stresses through the CRISPR-Cas system (Ricroch et al. 2017). For example, AB sensitivity in Arabidopsis was minimized through the addition of ABA-induced transcription repressors (AITR; adjust feedback of ABA), while declining AITR genes became nonfunctional (Tian et al. 2017a, b), leading to survival against abiotic stresses (drought and salinity) (Chen et al. 2021), while being less responsive (salinity) in the case of AITR5 gene overexpression (Chen et al. 2021). Furthermore, AITR6 also enhanced tolerance (Chen et al. 2021). Similarly, knocking out 3 genes (aitr2, aitr5, and aitr6) led to quintuple mutants resulting in enhanced resistivity towards stress (Bhattacharya et al. 2021).

Table 1 CRISPR/Cas induced resistance against abiotic stress
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9.1 Drought Stress

Plants get sensitive to stress as a result of unbalanced hormone levels, declining antioxidant actions, and higher ROS assembly (Abd El Mageed et al. 2023). Metabolic and signaling molecules build up, such as genes sensitive to low water along with transcription elements, resulting in enhanced plant survival against drought (Santosh Kumar et al. 2020). AREB1 overexpression makes plants resistant, while its removal makes plants sensitive to drought (Singh and Laxmi 2015). SlLBD40 (lateral organ boundaries domain transcription factor) knockout helps tomato plants against drought stress tolerance (Liu et al. 2020a, b). Furthermore, ARGOS8 gene increment declines ethylene activity, making Zea mays resistant to drought, leading to high grain yields (Hirai et al. 2007). WRKY3 and WRKY4 genes help in drought resistance, while WRKY transcription factors help against both stresses (Li et al. 2021a, b).

According to Chen et al. (2021), tomatoes are more resistant to water deficiency when the tomato Auxin Response Factor (SlARF4) gene is knocked down. By converting chromatin to a relaxed state, Arabidopsis histone acetyltransferase 1 (AtHAT1) encourages the activation of gene expression. By fusing the CRISPR/dCas9 gene with the Histone Acetyl Transferase (AtHAT) gene, Arabidopsis was shown to have improved resistance to drought stress (Roca Paixão et al. 2019). Negative regulators of drought tolerance include the genes for drought-induced SINA protein 1 (OsDIS1), drought and salt-tolerant protein 1 (OsDST), and ring finger protein 1 (OsSRFP1). Reducing the expression of these genes related to drought enhanced the levels of antioxidant enzymes, lowered H2O2 concentrations, and strengthened rice plants’ resistance to drought stress (Santosh Kumar et al. 2020).

Plants’ responses to dehydration and ABA signaling are controlled by the Enhanced Response 1 (ERA1) protein gene. Ogata et al. (2020) found that altering the OsERA1 gene in rice resulted in greater tolerance to drought stress. The mutant plants displayed increased sensitivity to ABA and stomatal closure under drought stress conditions.

9.2 Salinity Stress

Saline land will be increased in the future (up to 2025), which changes the plant physiology and morphology, leading to adverse changes (leaves shedding and spotting before their time, and severe ion disruption in cells) on plant growth and development (Dawood et al. 2022). Knocking out genes (AtWRKY3 and AtWRKY4) in A. thaliana causes more ion escape along with lower antioxidant actions, creating salinity sensitivity (Chen et al. 2021), while acquired osmotolerancegenes make plants saline tolerant (Kim et al. 2021).The GT-1 element’s regulatory role in rice led to the CRISPR/Cas9-mediated editing of the OsRAV2 gene, which demonstrated salt stress tolerance (Duan et al. 2016). Rice that was exposed to salt stress was resistant to CRISPR mutants that had lost the ability to function of SnRK2 and the osmotic stress/ABA-activated protein kinases SAPK-1 and SAPK-2 genes (Lou et al. 2017).

Other studies have developed CRISPR-mutants in rice to develop plants that can withstand salt stress by knocking out OsDST (Santosh Kumar et al. 2020), OsNAC45 (Zhang et al. 2020), AGO2 (ARGONAUTE2) (Yin et al. 2020), OsRR22 (rice type-B response regulator) (Zhang et al. 2019a, b, c), and OsBBS1 (bilateral blade senescenc1) (Zeng et al. 2018). Salt tolerance was increased in wheat plants with TaHAG1 gene knockout mutants produced by CRISPR/Cas9 (Zheng et al. 2021). Alam et al. (2022) produced rice mutant plants with altered DNA by focusing on the OsbHLH024 gene in order to investigate its function in conditions of salt stress. The osbhlh024 mutants had a single nucleotide base deleted, and when they were exposed to salt stress, their total chlorophyll content and shoot biomass increased significantly. Alfatih et al. (2020) reported that OsPQT3 knockout mutants in rice showed increased resistance to oxidative stress and salt stress, as well as elevated expression of OsGPX1, OsAPX1, and OsSOD1. Additionally, Mo et al. (2020) found that the loss of SLR1 functioned to promote mesocotyl and root growth, particularly in dark and salt stress conditions.

9.3 Heat Stress

Due to global warming, high temperatures are another limiting factor in crop reduction (Mohamed and Abdel-Hamid 2013). Using advanced tools, several genes (heat stress-responsive) have been detected to minimize intricate molecular system activities (such as the appearance of high heat sensing genes, indicator transduction, and metabolite assembly). Several mechanisms (heat shock proteins, heat shock transcription factors) enhance the pathway of signals beginning at high temperatures and decontaminate reactive oxygen species (Mohamed et al. 2019). CRISPR is a more powerful tool than others to create resistance against stresses (Duan et al. 2016). Irrespective of high temperature, SlAGAMOUS-LIKE 6 increases fruit set in tomatoes (Klap et al. 2017).

SlMAPK3 is a member of the mitogen-activated protein kinase family and plays a role in several environmental stress responses in tomatoes. Following genome editing with CRISPR/Cas9, slmapk3 mutants displayed increased thermotolerance in comparison to wild-type plants, suggesting that slmapk3 functions as a negative regulator of thermotolerance (Yu et al. 2019).

In the tomato apoplastic region ROS production is positively regulated by BRZ1, which also contributes to heat tolerance. This has been confirmed by bzr1 mutants based on CRISPR/Cas9, which demonstrated reduced H2O2 generation in apoplasts and decreased heat tolerance as measured by Respiratory Burst Oxidase Homolog 1 (RBOH1) (Yin et al. 2018).

Tomatoes that were created as CRISPR/Cas-mediated HSA1 (heat-stress sensitive albino 1) mutants were more sensitive to heat stress than plants of the wild type (Qiu et al. 2018). Li et al. (2017a, b) reported that thermosensitive male-sterile plants in maize were enhanced by CRISPR mutants of the thermosensitive genic malesterile 5 (TMS5) gene. In lettuce, knockouts of NCED4, a crucial regulatory enzyme in the production of ABA, allowed the seeds to germinate at a higher temperature. Consequently, in regions of production where temperatures are high, LsNCED4 mutants may have commercial value (Bertier et al. 2018).

9.4 Cold Stress

Metabolic trails and cell tissues get highly injured under cold temperatures (Jha and Mohamed 2023). Plants (inter-specific or inter-generic hybrids) have been modified against cold tolerance using traditional procedures. Certain negative genes (OsMYB30) that result in starch degradation and maltose amassing) were deleted by using CRISPR (Mo et al. 2020) to reduce cold sensitivity (Lv et al. 2017). Three genes (OsPIN5b, GS3, and OsMYB30) were also modified at once to cope with cold (Zeng et al. 2020). Annexin genes (OsAnn3 and OsAnn5) help to cope with cold at the seedling phase (Li et al. 2022).

To combat climate change and guarantee future food security, CRISPR/Cas9 is an alluring and approachable method for creating non-transgenic genome-edited crop plants (Mo et al. 2020). To improve the rice plant’s resistance to cold, editing is directed to eliminate a few negative regulator transcription factors. A transcription factor called OsMYB30 has a deleterious effect on cold tolerance by binding to the β-amylase gene promoter. According to Lv et al. (2017), OsMYB30 forms a complex with OsJAZ9 during cold stress, which suppresses the expression of the β-amylase gene and affects starch breakdown and maltose buildup. This could potentially lead to an increase in cold sensitivity.

OsPIN5b, GS3, and OsMYB30 were three genes that underwent simultaneous mutations by CRISPR/Cas9-based genome editing, exhibiting increased yield and resistance to cold stress (Zeng et al. 2020). Plant annexins are engaged in protecting plants from environmental stressors and controlling how they develop. At the seedling stage, the rice annexin genes OsAnn3 and OsAnn5 are positive regulators of cold stress resistance. Sensitivity to cold treatments was the outcome of knocking out OsAnn3 and OsAnn5 (Shen et al. 2017).

Low temperature (4 °C) was observed to boost SlNPR1 expression and protein concentration in tomatoes. CRISPR/Cas9-mediated genome editing of SlNPR1 induced the symptoms of chilling injury in tomato plants that were substantiated by the accumulation of hydrogen peroxide (H2O2), superoxide anion (O2 −), and malonic dialdehyde (MDA), as well as reduction in proline content, antioxidant enzymatic activities, and soluble protein content (Shu et al. 2023). In strawberries, overexpression of the FvICE1 gene led to phenotypic and physiological tolerance to cold and drought. On the other hand, strawberries with CRISPR/cas9-mediated gene editing displayed reduced resistance to cold and drought. According to Han et al. (2023), these findings implied that FvICE1 acted as a positive regulator of cold and drought.

9.5 Metal Stress

Linkage and gathering of heavy metals with oxidative stress led to cell damage, which could be minimized by crops using several techniques (Abu-Shahba et al. 2022). Such mechanisms are driven by multiple genes (Hasanuzzaman et al. 2019). Genes (OXP1 and γ-glutamylcyclotransferase) that build up high levels of glutathione create resistance to stress using CRISPR-Cas9. Cd resistance in plants (Arabidopsis) rather than WT Col0 was observed under oxp1/CRISPR mutant usage (Baeg et al. 2021).

For instance, the γ-glutamylcyclotransferase loss-of-function mutant exhibited protective features against heavy metal toxicity, indicating that the increased glutathione (GSH) accumulation in the loss-of-function mutants of OXP1 and γ-glutamylcyclotransferase demonstrates heavy metal and xenobiotic detoxification (Paulose et al. 2013). Understanding the role of a possible target, OsPRX2, for enhanced potassium deficit tolerance is another use of CRISPR-Cas-based editing in rice. Under K+ limitation, OsPRX2 has been shown to decrease ROS generation. It was discovered that stomatal closure and K+ deficit tolerance increase when OsPRX2 is overexpressed. Moreover, under the K+-deficiency tolerance, OsPRX2 deletion results in severe abnormalities in the phenotypic of leaves and stomatal opening (Mao et al. 2019).

In rice, the transporters OsNramp1, OsNramp5, and OsCd1 help the roots absorb cadmium from the soil. According to Chen et al. (2019a, b), OsHMA3 is responsible for sequestering cadmium within the root vacuole and adversely regulating xylem loading, while OsLCT1 is involved in cadmium transport to the grains. Reducing the amount of cadmium in grain crops has been partially achieved by genome editing techniques that manipulate the expression of these transporter genes. OsNramp5 and OsLCT1 CRISPR/Cas9-based mutations led to an acceptable cadmium level in rice (Wang et al. 2017). The primary route for Cs+ absorption and translocation in rice is the Cs + -permeable OsHAK1 transporter. Utilizing the CRISPR-Cas technology, transgenic plants devoid of OsHAK1 function were created in order to reduce the amount of radioactive caesium (Cs) that rice plants in Fukusima soil polluted with 137 Cs+ absorbed. According to Nieves-Cordones et al. (2017), the OsHAK1 knock-out plants showed remarkably lower levels of 137 Cs+ in their roots as well as lower radioactive caesium contents.

9.6 Herbicide Stress

Herbicide application is one of the best tools among others to minimize its population (Gage et al. 2019). While CRISPR-Cas9 edited genes to protect plants against weeds (Toda and Okamoto 2020), this led to a higher yield than crops grown by traditional procedures (Dong et al. 2021). Genes (acetyl-coenzyme A carboxylase and acetolactate synthase) editing (base) occurs against aryloxyphenoxy propionate-, sulfonylurea-, and imidazolinone-type herbicides using CRISPR in wheat (Zhang et al. 2020).

Tomato, soybean, rice, wheat, corn, watermelon, oilseed rape, tobacco, potato, and Arabidopsis are a few examples of crops that have been modified for ALS gene-based herbicide resistance (Dong et al. 2021). All things considered, genome editing provides a potent tool for creating crops resistant to herbicides by altering important genes like ALS. Through CRISPR-based editing of the OsALS1 gene, a novel herbicide tolerance phenotype has been inserted into Oryza sativa (Kuang et al. 2020).

According to research by Lu et al. (2021), large-scale genomic duplication or inversion can be designed in rice by CRISPR/Cas9 mediated genome editing, resulting in the development of new genes and characteristics. They demonstrated that leaves exhibited high transcript accumulation of CP12 and Ubiquitin2 genes, and that edited plants with homozygous structural variation alleles had 10 times higher expression levels of HPPD and PPO1, which led to herbicide resistance in field trials without compromising yield or other agronomically significant traits.

Herbicide-tolerant crop plants can be produced through genome editing using CRISPR/Cas9. Herbicide resistance was imparted in Solanum lycopersicum cv. Micro-Tom by CRISPR/Cas9-based targeted mutagenesis of three genes: EPSPS (5-Enolpyruvylshikimate-3-phosphate synthase), ALS (acetolactate synthase), and pds (phytoene desaturase) (Yang et al. 2022). These characteristics of herbicide tolerance present a potentially effective strategy for controlling weeds. The building of herbicide-resistant genes in crop plants could thus be precisely advanced by the CRISPR-based genome-editing technique.

9.7 Anoxia/Hypoxia Stress

Irrespective of oxygen usage by plants, its free form minimizes its yield and plants activate transcriptional factors along with initiating signals to cope stress (Zahra et al. 2021). ROS gathering (in rice shoots) awakens certain assemblies such as Lysigenous aerenchyma formed by Respiratory Burst Oxidase Homolog (Colmer and Pedersen 2008), and inside wheat and maize roots against stress tolerance (Yamauchi et al. 2017) due to prompted ethylene using CRISPR (Yamauchi et al. 2017).

10 Targeting the Structural and Regulatory Genes Enhancing Abiotic Stress Tolerance

Stress-related genes (especially structural genes) have a key role in stress detection. Several mechanisms (regulation of gene appearance, shielding from pathogens, and nitrogen fixation by ROS) have been described (Ribeiro et al. 2017). While several aberrations (male infertility, lower photosynthesis causing cell demise, and less production) occur due to overproduction of ROS due to stress (abiotic and oxidative) (Zafar et al. 2019), cell redox stability should be maintained through regular ROS production and scavenging checks (Abu-Shahba et al. 2022). Moreover, numerous T genes (coding antioxidant enzymes) are involved in foraging against stress survival (Abu-Shahba et al. 2022). While oxidative stress increases programmed cell death along with antioxidant decline through S genes (disturbs hormonal homeostasis), plant stress is sensitive (Zhao et al. 2017). SPCP2 and TaCP increase stress tolerance in crops (such as wheat and sweet potato), while others (Oryza sativa stress-related RING finger protein 1; negative regulator) increase H2O2 and minimizes antioxidant functions (Fang et al. 2015). Furthermore, tolerance is produced by OsSRFP1 knockdown (antioxidants get clearly adjusted and H2O2 disorders) (Fang et al. 2015).

Another gene class known as regulatory (giving code to transcription elements, kinases, and phosphatases) also initiates signals and manifestations of genes. Lower ROS-scavenging capacity and increased proline biosynthesis are caused by Arabidopsis S genes (NAM-ATAF1/2 and CUC2 TF), where ANAC069 binds to the core motif pattern in the promoter region (He et al. 2017). Similarly, its knockdown led to tolerance against stresses (salt and osmotic) (He et al. 2017). Table 2 shows several genes edited using the CRISPR-Cas tool in multiple crops.

Table 2 Gene editing occurred using CRISPR-Cas technology
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11 CRISPR-Based Targeted Gene Knockout for Abiotic Stress Tolerance

Multiple genes could be edited using CRISPR (rather than new gene detection using genome checking) to develop stress-resistant species (Abdelrahman et al. 2018). For example, spindly gene (SPY-3) knockouts create stress tolerance (less water and high salt conditions). Arabidopsis, while MODD and TaDREB2 work depressingly (Kim et al. 2018). MicroRNAs (20–22-nucleotide noncoding RNAs target transcription factors) should be revised using CRISPR for resistive crop development (Farhat et al. 2019), especially fine-tuning the appearance of miRNA (Tang and Chu 2017).

12 CRISPR/Cas9 Uses in Considering Abiotic Stress Tolerance

The role of CRISPR/Cas9 (gene editing) has been observed in various species (Lei et al. 2014), using improved sgRNAs (for gene detection) (Montague et al. 2014). Practices (for targeted mutagenesis) (Shan et al. 2014), along with required paths and accessories (Xing et al. 2014), accessible through AddGene (a volunteer plasmid storehouse) (https://www.addgene.org/crispr/plant/), helpful in producing tolerant crops against abiotic stress, have been mentioned. Various genes and the collaboration of various mechanisms (indicating, adjusting, and metabolic ways) led to abiotic stress (Mickelbart et al. 2015).

Complete genome replication mechanisms along gene segment efficient severance have been undertaken by plants, which could be resolved through CRISPR-Cas9 (sgRNAs; targeting various genes), because sole gene editing is not enough for the required results (Zhou et al. 2014). Gene marking through HDR (another method) has a role in stress reactions; using such practices, useful genes could be fired and various genes could be interpreted (for abiotic stress). Plants could be developed in the presence of high native germplasm and gene deviations while becoming difficult due to the absence of the mentioned components, which could be resolved by gene editing (using sgRNA for multiple gene pointing) on an advanced genetic level. The role of CRISPR in humans (vigorous gene assortment) has also been recorded (Wang et al. 2014).

Discovery of other Cas9 kinds will enhance CRISPR dominancy, such as dCas9 (using CRISPRi), which could interrupt genes workability (Qi et al. 2013), and improve transcriptional subjugation (due to KRAB/SID binding with dCas9) (Konermann et al. 2013), which led to considerable improved mutagenesis (Cho et al. 2014). It also plays a role in other activities (methylation, chromatin positions, controlling or polishing genes). Cas9 rearrangement is also helpful in accurate editing such as TALENS production (using CIB1 and CRY2; light inducible domains) (Konermann et al. 2013).

Specific genes have been improved through various sgRNAs (Perez-Pinera et al. 2013). The efficiency of pointed gene transcription has been controlled by CRISPR (activator or repressor) (Piatek et al. 2015). Non-required results could be skipped using uncertain promoters (initiative of Cas9 and sgRNA appearance). AP2/ERF (transcriptive gene) and ABA-INSENSITIVE4 produce sudden changes (using ZFNs in combination with heat shock protein led to the required results) (Osakabe et al. 2010). Homozygous biallelic mutants could be characterized by CRISPR tools (Kim et al. 2014), along with homozygous transgenic plant production (the quickest approach led to stress-tolerant species production) (Feng et al. 2013; Zhou et al. 2014). Using nucleases (via transgenic procedures) is helpful in improving plant production (known as non-GM) (Marton et al. 2010). Figure 7 represents the role of CRISPR for improved crop production against abiotic stress tolerance.

Fig. 7
figure 7

Action of the CRISPR-Cas9 method against stress survival using efficient genomics

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13 Proofs of Multiple Genes Modification in Various Crops Through CRISPR-Cas9

Table 3 shows a particular gene pattern assessment using the CRISPR-Cas9 tool. Various studies have clarified CRISPR-Cas’s role in tolerant crop development towards abiotic stress. SAPK2 (ABA-activated protein kinase 2) features were explained by avoiding mutants (produced by CRISPR) having a high role in abiotic stress (Lou et al. 2017). slmapk3 mutants were produced (through CRISPR) by pointing MAPKs while comparing remote and transgenic tomatoes (towards stress), where higher symptoms (wilting, more membrane injury, and hydrogen peroxide, with fewer antioxidant enzyme actions) were noted in transgenic tomatoes than wild-type tomatoes. Similarly, CRISPR-Cas9 developed ARGOS8 variants for higher grain production under water scarcity (Shi et al. 2017). Furthermore, ARGOS8 variants (addition of the GOS2 promoter to the 5’-untranslated section of such a gene) having high transcripts in relation to the native allele, as noted in all maize tissues, led to higher yields and improved crop production under abiotic stress.

Table 3 Overall assessment among the particular genome editing elements
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14 Confrontation and Prospects in Developing Stress Tolerant Crops Through Moderating Genes Using CRISPR-Cas Tools

Various factors (less effective entrant genes, significant alteration, and plant restoration tools) limit the role of CRISPR-Cas in abiotic-tolerant crop species, which could be overcome by several methods (detecting new genes and accurate plant tissue culture procedures). With the passage of time, more advanced genetic tools (for improved drought-tolerant crop production) have been used (Mickelbart et al. 2015). Multiple mechanisms (depicting dictation elements and molecular division of indicating paths) are responsible for the discovery of new genes (Licausi et al. 2013). Through CRISPR-Cas9, various stress-tolerant genes have been identified in multiple crop species (Grover et al. 2013), using multiple tools (i.e., promoter recognition to initiate and prompt Cas9 along diverse bright correspondents, and assortment pointers) (Bhowmik et al. 2018). For traits (responsible for abiotic stress tolerance), detection, workability, and categorization along gRNAs could be done using microspore-based gene editing systems rather than typical body cell-based gene editing (Bhowmik et al. 2018).

Besides being a useful and effective tool (CRISPR), it has some issues, such as off-target editing (unnecessary genes edited along essential genes) through an effective blend of NGG PAM and definite gRNA (Zhang et al. 2015), while NAG PAM also has a fusion with Cas9 (Hsu et al. 2013). The involvement of various nucleases makes CRISPR unable to cut the whole necessary pattern (due to regulated PAM). Similarly, there is a lack of suitable procedures to provide lots of giver patterns at the DSB point using HDR-mediated accurate editing. CRISPR is a friendly tool to develop crops that are more tolerant (more yielding, more climate-hardy) against abiotic stress to resolve food decline.

15 Modifying Genes to Cope with Abiotic Stress Via CRISPR-Cas9 System

15.1 Stress Signal Transduction

In order to withstand abiotic stresses, plants react to various outsider incentives, such as transportation by the cytosolic kinase domain, as a result of signals detected by multiple membrane-localized protein receptors (at the intracellular level) (Osakabe et al. 2013). Numerous (600–1000) RLKs (stress-inducible receptor-like kinases) with multiple actions have been found in the growth and design of crop species (A. thaliana and O. sativa), while also detecting inner and outer incitements (connecting hormonal and ecological signaling) through various procedures. For example, ABA signaling has certain RLKs (CRK36, PRK1, GHR1, PERK4, ABA, and osmotic-stress-inducible receptor-like cytosolic kinase 1) in Arabidopsis during waterless situations (Hua et al. 2012). Hormone incentives to cope with abiotic stress are also described by HKs (histidine kinases) (Nongpiur et al. 2012), where phytohormone incentives are carried by their multiple members (phytohormones like cytokinin and ethylene) (Pham et al. 2012). HKs and RLKs downstream detect stress signals and activate multiple essential hormones in response, where MAPK (signaling pathways) make species tolerant to stress, such as NPK1 (Nicotiana-Protein-Kinase1) in maize towards low temperature stress.

15.2 Transcription Elements

Dehydration-responsive element-binding-factors (DREB1A, DREB2A, and DREB1B) attach to cis-acting components (situated at the promoter point of the gene causing tolerance) to control gene action. The competence of DREB could be enhanced via CRIPSR/Cas9 to cope with abiotic stress. Abscisic acid (ABA) abating makes bZIP (leucine-zipper) indestructible when withstanding stress. Furthermore, ABA signaling in A. thaliana is controlled by multiple components (AREB1, AREB2, and ABF3) during waterless conditions, which could be advanced through CRISPR usage (Yoshida et al. 2010).

15.3 Phytohormones Metabolic Pathway

Phytohormones (less quantity in cells) have a great role in various actions of plants (adaption, regulation of growth and development, nutrition distribution, and conversion of bases) towards different conditions (Fahad et al. 2015). They are very important signaling molecules for plants (sessile organisms), which control certain responses (molecular and physiological). Waterless conditions become ineffective in the presence of Phaseolus vulgaris PvNCED1 in transgenic tobacco (Qin and Zeevaart 2002). Similarly high rice grain yield in the presence of ABA3/LOS5 (during drought situations) and also less leaf ageing in the presence of more cytokinin (Ma 2008), followed by lower leaf ageing due to flooding in Arabidopsis due to isopentenyl transferase (IPT) (Huynh et al. 2005).

16 Role of CRISPR-Cas System in Developing Climate Smart Crops

Finding specialty and editing any target gene pattern of plants to create target mutations based on DNA or RNA could be easily and efficiently done by CRISPR-Cas9 technology, but it needs to be improved to increase plants heritability. Where unnecessary genes could get targeted, this led to stress responses in the case of Cas9, which could be lessened using appropriate sgRNAs (which only edit target genes), such as CRISPR-P usage in various plant species (Jain 2015). Cpf1 (in mammalian cells) causes mutagenesis (Mahfouz 2017) by working (multiple cleavage at a pointed site) in the presence of crRNA (using T-rich PAM with a RuvC-like nuclease area rather than HNH) and leading to DNA cleavage a little far from PAM. Cpf1 is target-specific and more efficient in plants than animals (Tang et al. 2017a, b).

17 Advantages of Genome Editing Over Other Conventional and Transgenic Techniques

Specific features from one line into another are inserted using a typical breeding cross (repeated), while mutation breeding causes arbitrary changes that take 8–12 years. Similarly, transgenic breeding (unsystematic assimilation of distant DNA in the genome) also transmits beneficial genes within 2–5 years without continual backcrossing. Furthermore, gene editing causes less alteration in DNA (which looks natural) for developed features than external DNA assimilation for resilient crop development in less time. Gene editing only edits pointed gene sites effectively (Song et al. 2016) and with precise DNA editing (Barrangou 2015).

18 Limitations

Although the CRISPR/Cas systems exhibit powerful ability in crop genetic improvement, some limitations still need to be overcome in this field. SpCas9 requires a 50-NGG-30PAM immediately adjacent to the 20 nt DNA target sequence because it can only recognize NGG PAM sites, which limits its effectiveness. Although this restriction is vital, the goal is to turn off the gene through selective mutagenesis in any situation (Fig. 8). Agrobacterium tumefaciens-mediated transformation is necessary to use the CRISPR/Cas system in plants, however it is a time-consuming, expensive, and difficult process. Target gene options are extremely constrained. On the one hand, shutting down a gene by itself cannot confer resistance because the role of resistance genes is redundant. On the other hand, PAM limits the knockout of resistance genes, thus sequences near PAM must be chosen. The plant genome may experience random off-target changes due to CRISPR/Cas. The inability to properly isolate Cas proteins and produce transgene-free crops has hampered the commercialization of CRISPR-edited crops (Wang et al. 2022). Currently, homologous donor sequence transformation into plant cells has a low efficiency and the homologous recombination route (knock-in/gene replacement) has a lower efficiency, which reduces the difficulty and efficiency of knock-in. Therefore, there is still much work to be done before CRISPR/Cas-mediated homologous recombination in plants can effectively knock in genes (Wang et al. 2022).

Fig. 8
figure 8

Limitations of the current CRISPR/Cas system

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Varietal usage has been recorded for CRISPR-Cas9, and currently CRISPRi (balancing RNAi) is doing gene quieting (Qi et al. 2013). CRISPR works better when paired with other tools, such as ease of feature analysis along molecular crossing (which occurs through genetic alteration at the allelic level) and wrong gene correction (Zaidi et al. 2016). It also lowers the herbicide (chemical) application on plants through gene editing (Xu et al. 2015). CRISPR should be used with care for the required results (Zaidi et al. 2016), due to its large size and the lack of unnecessary PAM site points caused by RNA misguidance (Zaidi et al. 2020). Viruses (with speedy replication) do not get affected by CRISPR. Various states don’t allow CRISPR created crops due to social problems (Eş et al. 2019).

19 Conclusions

CRISPR-mediated editing has the potential to improve crops with abiotic stress tolerance and improved nutritional content. The CRISPR-based genome editing tool is considered one of the most powerful technologies for improving agriculture to feed the rapidly growing population. It can develop genome-edited crop varieties with no foreign-gene integration like those created through conventional breeding. Gene editing is a very powerful method rather than other traditional breeding procedures due to the accurate and minute alteration production in plant DNA (without analyzing external DNA) for crop improvement, high yield, resistance to stress, and vice versa. Climate-hardy crops could be obtained in less time than 12–15 years (without repeated crossing). CRISPR only targets specific points, not whole genome patterns, resulting in accurate mutagenesis.

CRISPR has made crops tolerant to abiotic stress by editing genes irrespective of their availability, which will be a powerful tool to be applied on a commercial level. This technology should be used in the agriculture sector for better results, although its limitations are there.. In the future, researchers will find CRISPR technology more profitable to use in multiple sectors. The global population needs to be fed as climate change has severely affected food security, which could be overcome in the future through advancements in CRIPSR technology.