Muhammad Ahtisham 
and Zainab Obaid
University of Agriculture, Faisalabad, Pakistan 
Correspondence to: Muhammad Ahtisham,,ahtishamislam10@gmail.com
Additional information
- Ethical approval: N/a
- Consent: N/a
- Funding: No industry funding
- Conflicts of interest: N/a
- Author contribution:Muhammad Ahtisham and Zainab Obaid – Conceptualization, Writing – original draft, review and editing
- Guarantor: Muhammad Ahtisham
- Provenance and peer-review:
Commissioned and externally peer-reviewed - Data availability statement: N/a
Keywords: Abiotic stress tolerance, CAS9, Climate change adaptation, CRISPR, Crop resilience, Genetic modification.
Peer Review
Received: 21 October 2024
Accepted: 28 October 2024
Published: 11 November 2024
Abstract
Climate change is posing a serious threat to future food security worldwide. With changing climate patterns, it has become increasingly urgent to utilize innovative technologies to make crops more resilient to climate changes. CRISPR/CAS9 has emerged as an innovative tool for the genetic modification of crops for improvement against climate change and stresses related to it. The review highlights the use of CRISPR/CAS9 to make staple crops such as wheat, rice, maize, barley soybean, and tomatoes more resilient against climatic stresses of temperature, salinity, and drought. By utilizing CRISPR techniques of gene knockout and knockdown, scientists are identifying potential genes that can help to make crops resilient to climate change in the future.
Moreover, in addition to the identification of the gene, CRISPR/CAS9 is enabling the researcher to understand the role of different genes under climate stress. By understanding the mechanism of gene action, researchers and plant breeders can work more precisely and efficiently by focusing on the gene relevant to make crops more resilient to climate change. Although CRISPR/CAS9-mediated crop improvement is not employed in most crops, a significant amount of work is still done in crops such as rice, soybean, and other crops discussed in the review. The versatility, precision, and ease of use enable scientists to edit or regulate multiple genes simultaneously in a crop. In short, although climate change is posing a threat to our future, food security tools like CRISPR/CAS9 are also equipping scientists with abilities to tackle these challenges.
Introduction
CRISPR/Cas9 is an advanced and promising technology in terms of targeted editing of the genome of crop species.1 It is a robust biotechnological tool that can be used for replacement, modification, knockout, and regulation of gene expression at the genomic level.2 CRISPR and other genome editing tools have become indispensable for the rapid improvement of traits for tolerance against abiotic stresses.3 In contrast to other methods of crop improvement, genetically modified organism are often a much better option under given circumstances to ensure a sustainable food supply for future generations.4 By 2050, the global population is expected to reach 10 billion. In the past, major crops were able to meet the food demands of a growing population, but the current rate of population growth means that the improvement of yield in major crops such as wheat, rice, and maize is not sufficient to meet the ever-increasing food demand.5 Climate change, shifting patterns of rainfalls, and ever-increasing temperatures are real threats to the agricultural sector, and genetic modification offers promising solutions to these problems. The yield of crops such as vegetables and legumes has declined by 37.7%, owing to a 50% decline in water availability and a 31.5% decline due to an increase of 4°C above the baseline of 20°C.6 With an increasing population and decreasing agricultural land, under the threat of climate change, the practical solution for food security is the development of climate-smart/resilient crop varieties.7
Transgenic technologies have played a crucial role in the improvement of yields, reductions of CO2 emission, lower cost of production, and resistance to insecticides and weedicides, and there are 525 transgenic events in 32 crops that are under cultivation with approval.8 CRISPR/CAS9 is an emerging technology with user-friendly tools that can help us tackle the challenges of food security by developing transgenic or even nontransgenic crop varieties.9 Since the introduction of the CRISPR/CAS 9 system, it has been extensively used to modify the expression of genes in different crops. The aims of the study were to highlight the importance of CRISPR/Cas9 in improving crops by enhancing crops’ resilience to climate change.
CRISPR/CAS 9 How It All Works
CRISPR/CAS9 identifies the specific site in the genome with the help of the guide RNA (gRNA), this recognition is dependent upon the complementarity between the gRNA and the target sequence in the genome, and this recognition is also aided by the presence of protospacer adjacent motif near the recognition site; once CAS identifies the site in DNA, it creates a double-stranded break (DSB) in the plant.10 Once a DSB is created, the plant activates its repair mechanism, using nonhomologous end-joining (NHEJ) to the heel, leading to small deletion or insertion in DNA, consequently resulting in gene knockout.11 In contrast to this, homology-directed repair can be used to replace or insert a new template sequence at the site of cleavage.12 In this way, CRISPR/CAS9 can be used to modify the action of the undesirable genes (with the help of NHEJ) and can also be used to introduce favorable traits in plants such as climate resilience, disease resistance, and improvement of yield.13 This ease of using CRISPR/CAS makes it a very helpful tool for plant breeders to improve crops in comparatively small duration without long cycles of plant selections for improvement (Figures 1 and 2).14
Attribution: Mariuswalter, translation by The Bartgry, via Wikimedia Commons.
Attribution: CC BY-SA 4.0, via Wikimedia Commons.
How CRISPR/CAS9 Is Making Crops More Resilient to Climate Change
Rice (Oryza sativa)
Due to its environmental resilience, rice is cultivated as a staple crop all over the world, feeding almost 50% of the world’s population. However, biotic and abiotic stresses due to climate change are real problems for rice production, and with the advancement of tools such as CRISPR/CAS9 and their ease of use, they are becoming a possible solution to overcome the problems associated with rice production.15 The osNAC006 gene in rice plays a crucial role in heat sensitivity and drought tolerance by regulating the expression of the gene involved in responding to these stimuli, the knockout of the OsNAC006 gene using CRISPER/CAS9 developed drought and heat sensitivity in rice plants, and this phenomenon can be used in manipulating heat and drought tolerance for the future improvement of crops.16 Another study that observed the effect of knockdown of the OsANN3 gene in rice resulted in sensitivity to drought, indicating the role of the gene in drought tolerance.17 Cold stress is one of the major problems in subtropical and tropical regions of rice cultivation.18 The CRISPR/Cas9 technology helps to study the role of genes by analyzing the phenotypes of mutants produced using CRISPR. The OsPRP1 gene plays a vital role in cold tolerance in rice by enhancing the activity of antioxidants and by maintaining the cross talk of signaling pathways in rice plants to cold stress, downregulation of OsPRP1 in rice using CRISPR/CAS9 resulted in the development of cold sensitivity in rice mutant plants, and it indicates that the gene can be exploited for the future improvement in rice for cold tolerance.19
The knockdown of the OsAnn3 gene using CRISPR/CAS9 in rice resulted in a sensitivity of the mutant line to cold, assuring the role of the gene in cold stress.20 Salt tolerance in rice was improved by the knockout of the OsRR22 gene using CRISPR/CAS9 in rice plants.21 Similarly, salt tolerance was created in mutant lines of rice by using CRISPR/CAS9 by inducing a deletion mutation of a single nucleotide in the OsRR22 gene, and salt-tolerant lines were identified as mutants exposed to salt stress.22 OsNAC041, a transcription factor in rice plants when mutated with CRISPR/CAS9 mutant plants, improved salinity tolerance and increased height.23 Targeted mutation of the OsOTS1 gene produced plants that were sensitive to salinity tolerance, indicating the major role of the OsOTS1 gene in tolerance against salinity stress.24 Another drought and salinity tolerance OsPUB7 gene in rice was edited using a CRISPR/CAS9 vector, and mutant lines were developed; then, mutant lines were found drought and salt tolerant.25
Wheat (Triticum aestivum)
Wheat ranks among the top three cereals all over the world, with an annual production of 700 million tons.26 It is a widely grown crop cultivated on 217 million hectares of land annually. About a fifth of the world’s population is relying on wheat for its source of food calories and carbohydrates which shows its great importance in human nutrition.27 Like all other crops, wheat also poses a great deal of threat due to increasing global temperatures, changes in precipitation, and increasing frequency of extreme weather.28 It is estimated that a 1-degree Celsius increase in temperature can cause up to a 10–20% decrease in overall yield globally.29 Sensitive stages like flowering, anthesis, and milking stage are highly affected by extreme temperature changes which affect wheat yield, grain weight, and size.30 Luckily, CRISPR/CAS offers solutions to these adverse effects, knocking off the LTP gene with the use of CRISPR/CAS has proven effective in increasing drought tolerance in wheat.31 Editing of the TaSBElla gene has not only boosted grain yield in drought conditions but also enhanced starch composition.32 The SgRNA CRISPR/Cas9 genome editing system was used to deactivate five homologous genes TaSal1 in wheat. Lines showing complete knockout of genes were identified, and the mutated plants showed better growth on polyethylene media than wild-type seedlings. Research suggests using the same system to induce mutation in TaSal1 genes in hexaploid wheat varieties.33
Maize (Zea mays)
Maize, among wheat and rice, plays a crucial role in providing food worldwide. As a major crop in many countries, it becomes an essential crop for industries, as it is grown in rain-fed areas mostly. However, climate change, water scarcity, and erosion in these areas are posing a threat to future yields of maize.34 The CRISPR/CAS9 technology is revolutionizing maze breeding and helping to overcome the effects of climate change. A recent study has shown the use of CRISPR/CAS9 to improve the yield of maize under drought conditions by introducing a novel GOS2 promoter using an advanced CRISPR/CAS technology in the 5-untranslated region of the ARGOS8 gene in maize, and mutants showed increased grain yield of 5 bustles per acre under drought condition.35 Unfolded protein and heat shock protein responses (UPR and HSR) are the defense systems of the plants in response to heat stress, knockdown of the transcription factor bZIP60 prevented the upregulation of the heat shock protein when exposed to heat stress.36 This study showed how maize plants behave at the molecular level under heat stress with the help of the knockdown of transcription factors.
A study has shown that the endoplasmic reticulum stress response factor ZmPP2C-A gene is negatively linked with drought tolerance in maize, and knockout of the gene ZmPP2C-A resulted in improvement against drought stress in maize plants and research suggested the use of mutants for improvement in maize breeding for drought stress.37 Another gene ZmHKT1 was identified to be linked to salinity tolerance in maize, it was found in the study that knockout of the gene using CRISPR/CAS9 increased the concentration of salts in plants, and plants became hypersensitive to drought stress.38 CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) genes in maize are found to be directly responsible for the yield, and CRISPR Cas9 genome editing was used to target CLE genes that increased grain-related traits in maize.39
Barley (Hordeum vulgare)
Barley is the most adaptable cereal and ranks fifth among the cereal crops in terms of the area of production and quantity produced. Barley also has good resistance to dry heat as compared to other cereals.40 HvCKX1 and HvCKX3 genes were knocked out by using the CRISPR RNA-guided system and mutant lines were generated; the observed results showed that single CKX gene knocking out was not enough to boost grain yield in barley even though root morphology advancement like increased surface area, greater length, and more number of root hair were detected in the mutant KO lines.41 A study has shown that the TaHsfA6b gene transferred from wheat into barley alters other gene expressions and consequently develops barley thermos tolerance.42 A study has identified cold tolerance genes in barley on Fr-QTL and identified two components found factor (CBF) genes most relevant to cold stress. Moreover, the study suggested the introduction of the identified cold tolerance genes in favorable genotypes.43 However, not a considerable amount of work is done on barley with the help of CRISPR/CAS9 to make it climate resilient. Still, many potential genes for environmental resilience have been identified, and their expression can be manipulated using the CRISPR/CAS9 technology. Research found a higher level of expression of genes HvHspc70-4, HvHspc70-N, HvHsp70Mt702, HvHsp100-2, HvHspc70-5a, HvHspc70-5b, HvHspc70-N2, and HvHsp110-3, HvHsp90-1, and HvHsp100-1 in barley when exposed to heat stress.44 These genes can be potential candidates for future improvement of barley to make it resilient to climate change using CRISPR/CAS in the future.
Soybean: (Glycine max)
Soybean is one of the most important crops worldwide.45 It is considered one of the largest sources of animal protein and vegetable oil.46 Many genes have been identified in soybeans to make them more resilient to climate change stresses using CRISPR/CAS9. Drought stress is one of the most devastating stresses in soybean, sometimes affecting yields up to 40%.47 In a study, it was found that the knockout of gene GmNAC8 using CRISPR in soybeans resulted in the sensitivity of mutant lines to drought stress, showing the positive contribution of the gene in drought tolerance.48 The knockout of the gene GmNAC12 in soybeans using CISPR/CAS9 resulted in a decrease in the survival rate of the mutant line by 46%, showing the importance of the gene in regulating drought tolerance in soybeans.49 In a study, it was found that knockdown of the gene gmhdz4 in soybean using CRISPR/CAS9 improved drought tolerance in mutant lines by improving osmolyte accumulation, root growth, and antioxidant properties of the plant.50 The overexpression of the gene GmCAMTA12 facilitated by the CRISPR gene editing resulted in enhanced drought tolerance and overall growth of mutants under drought conditions.51
Mutation of the GmAITR genes in soybeans using CRISPR led to significant improvement in salinity tolerance in soybeans.52 It was observed that CRISPR-mediated knockout of the gene GmHsp90A2 in soybean made the mutant lines sensitive to heat stress while the line showing overexpression of the gene became more heat tolerant, and the gene can be used to make soybean resilient to heat stress in the future.53 Knockout of the gene GmMYB118 resulted in soybean plants becoming more sensitive to reduced salinity tolerance. In contrast, plants with overexpression of the gene exhibited increased tolerance to salinity and drought stress.54 In another study, CRISPR/CAS was used to knock out the GmNHX5 gene in soybeans, and mutant plants showed reduced levels of salinity tolerance in contrast to the overexpression line.55 CRISPR-mediated gene knockout was used in the identification of another gene for salinity stress tolerance in soybean, as the mutant plant with knocked out NAC domain transcription factor, GmNAC06, showed sensitivity to salinity stress, while in contrast, overexpression lines showed increased proline, glycine levels, and improved ratios of Na+/K+ to tackle with salinity stress.56
One very special study showed that the role of the genes GmPHYA2/E4 and GmPHYA3/E3 is involved in the delay of flowering in soybeans, and in the future, these genes can be used to induce earliness in soybeans which can help in making soybeans more resilient in future.57 All these studies are already giving a direction to the researchers about the use of CRISPR/CAS9 technology, especially its role in the identification of favorable genes to make soybeans more climate resilient.
Tomato (Solanum lycopersicum)
Tomato is known for its health benefits and high nutritive content, being rich in antioxidants, saturated fats, and sodium. It is a well-recognized vegetable crop and an important part of every cuisine worldwide.58 Mitogen-activated proteins (MAPKs) genes respond to drought stress, and CRISPR Cas9 was used to generate SIMAPK3 gene mutants in tomato lines that resulted in drought response in the mutant lines by protecting cell membrane from oxidative damage and transcription of stress-related genes.59 When the SINPR1 gene was knocked out in tomato mutants by CRISPR, it showed higher drought sensitivity as it increased stomatal aperture, higher electrolyte leakage, and lower levels of antioxidant enzymes; the observed changes prompted the importance of the targeted gene.60
It is reported that lower gibberellin activity (GA) helps tomatoes to retain water content by decreasing leaf surface area and stomatal conductance, and a single GID1 gene was inhibited, and low GA activity in the mutants lowers the transpiration rates as it reduced leaf expansion, stomatal closure, xylem proliferation which helps it adapt better in drought and water-scarce conditions.61 The SlLBD40 gene which belongs to LATERAL ORGAN BOUNDARIES DOMAIN (LBD) was found to be a negative regulator of drought in tomatoes, and the KO lines of SILBD40 showed increased drought tolerance in the mutant line.62 Thermotolerance in tomatoes can be influenced by the BRASSINAZOLE RESISTANT 1 (BZR1) gene which is a regulator of brassinosteroid, the overexpression of BZR1 enhanced heat tolerance by increasing the production of H2O2.63 CGFS-types (SlGRXS14, SlGRXS15, SlGRXS16, and SlGRXS17) with multiplex CRISPR/Cas9 systems were targeted, and results showed that SLYGRXS has particular roles against abiotic stress which can help develop mutant lines resistant to climate change.64
ARF4 (auxin response factors) were inhibited by CRISPR (knocked out), and it revealed that the downregulation of SLARF4 enhanced the mutant’s ability to tolerate salt and osmotic stress by promoting root development, increasing soluble sugar, maintaining chlorophyll levels, and regulating ABA-related genes.65 Another key regulator SIHAK20 was discovered that it is directly responsible for Na+/k+ ion regulation, and knockout mutants showed increased sensitivity to salt stress.66 The hybrid proline-rich protein-1 (HyPRP1) gene was identified and suppressed by using CRISPR as it is a negative regulator of salt stress in tomatoes, and the KO lines showed high salinity tolerance.67 The SIABIG1 gene, a member of the HD-ZIP II transcription family, is another negative regulator of salt stress, and its KO line has the ability to develop salt-resilient tomato variety.68 SP5G-SELF-PRUNING 5G is a flowering repressor gene that can be induced due to long days which inhibits flowering, and CRISPR-mediated edited mutations lead to early rapid flowering beneficial for tackling changes in photosensitivity due to shifts in climatic patterns.69 Highly conserved CBFs have an important role in chilling stress, and CRISPR-mediated SLCBF1 KO mutants proved this when they showed increased sensitivity to chilling stress.70 Knocking out of the SLUVR8 gene (UV RESISTANCE LOCUS 8) in tomatoes helps in seedling development in UV-B stress conditions.70
Other Vegetable Crops
Lettuce (Lactuca sativa L.) is popular globally for its dietary contents due to its low calories, fats, sodium, and high fiber contents and is also a great source of bioactive compounds.71 In lettuce, LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4) is the gene responsible for the germination of seeds to germinate under high temperatures. The precision and stability of CRISPR/Cas9 mutations were also calculated in this experiment, and the RNA-guided method was more stable. Knocking out of the LsNCED4 gene also allowed to bypass the term “inhibition” controlled by it and the seed germinated at temperatures as high as 37°C, this was a successful experiment concerning building climate-resilient crops with the help of CRISPR-gene editing.72
Pumpkin salt tolerance is linked with the RBHOD gene that controls hydrogen peroxide H2O2 accumulations. To investigate its CRISPR/Cas9, gene editing was used and RBHOD was targeted, the KO mutants led to lower H2O2 and decreased potassium (K+) uptake in root apex, resulting in salt intolerance. These results show the importance of the RBHOD gene and show its overexpression can lead to increased salt tolerance in pumpkins.73 CRISPR/Cas9 was also successful in editing specific genes in ground cherry (Physalis pruinosa) to improve growth, fruiting flowering, and other production traits. Ppr-AGO7, Ppr-SP5G, and Ppr-CLV1 were targeted, 24% increase in fruit mass, more floral growth, and higher concentration of fruits were observed which dramatic increase in yield and quality of the crop, which is necessary to tackle the decreasing yields due to the climate change (Table 1).
| Table 1: Summary of All Genes of Crops Discussed in the Study and CRISPR/CAS9 Techniques Applied to it. | ||||
| Crop | Gene | Targeted Trait | Modification | Result |
| Rice | OsNAC006 | Heat and drought tolerance | Knockout | Sensitivity to drought and heat |
| Rice | OsANN3 | Drought tolerance | Knockdown | Increased drought sensitivity |
| Rice | OsPRPI | Cold tolerance | Downregulation | Cold sensitivity developed |
| Rice | OsRR22 | Salt tolerance | Knockout | Improved salt tolerance |
| Rice | OsNAC041 | Salinity tolerance | Mutation | Enhanced salinity tolerance |
| Rice | OsOTS1 | Salinity tolerance | Targeted mutation | Significant role in salinity tolerance |
| Rice | OsPUB7 | Drought and salinity tolerance | Gene editing | Developed tolerance to drought and salt |
| Wheat | LTP | Drought tolerance | Knockout | Increased drought tolerance |
| Wheat | TaSBElla | Grain yield and starch | Editing | Enhanced yield under drought |
| Wheat | TaSal1 | Growth under drought | Knockout | Improved growth under stress |
| Maize | ARGOS8 | Grain yield under drought | Gene editing | Yield increased by 5 bushels/acre |
| Maize | bZIP60 | Heat stress response | Knockdown | No upregulation of heat shock proteins |
| Maize | ZmPP2C-A | Drought tolerance | Knockout | Improved drought resistance |
| Maize | ZmHKT1 | Salinity tolerance | Knockout | Increased drought sensitivity |
| Maize | CLE | Yield-related traits | Gene editing | Enhanced grain traits |
| Barley | HvHsp70 | Heat tolerance | Expression analysis | Increased heat tolerance genes |
| Barley | TaHsfA6b | Thermotolerance | Transferred gene | Enhanced thermotolerance |
| Barley | CBF genes | Cold tolerance | Identification | Potential candidates identified |
| Soybean | GmNAC8 | Drought tolerance | Knockout | Sensitivity to drought |
| Soybean | GmNAC12 | Drought tolerance | Knockout | 46% decrease in survival rate |
| Soybean | gmhdz4 | Drought tolerance | Knockdown | Improved root growth and stressresponse |
| Soybean | GmCAMTA12 | Growth under drought | Overexpression | Enhanced drought tolerance |
| Soybean | GmAITR | Salinity tolerance | Mutation | Improved salinity tolerance |
| Soybean | DrB2a | Drought and salinity tolerance | Knockout | Enhanced tolerance to stresses |
| Soybean | DrB2b | Drought and salinity tolerance | Knockout | Increased stress tolerance |
| Lettuce | LsNCED4 | Heat tolerance | Knockout | Enabled germination at 37°C |
| Pumpkin | RBOHD | Salt tolerance | Knockout | Reduced H2O2 accumulation better tolerance |
| Groundcherry | Ppr-AGO7 | Leaf and floral organ development | Gene editing | Narrower leaves and petals |
| Groundcherry | Ppr-SP | Flowering and plant architecture | Gene editing | Enhanced yield and architecture |
| Groundcherry | Ppr-SP5G | Flowering time and day length sensitivity | CRISPR targeting | Modified flowering response |
| Tomato | SlMAPK3 | Drought response | Knockout | Protected cell membranes from oxidative damage |
| Tomato | SINPR1 | Drought sensitivity | Knockout | Increased stomatal aperture and electrolyte leakage |
| Tomato | GID1 | Water retention | Knockout | Lower transpiration rates and reduced leaf expansion |
| Tomato | SlLBD40 | Drought tolerance | Knockout | Increased drought tolerance in mutant lines |
| Tomato | BZR1 | Heat tolerance | Overexpression | Enhanced heat tolerance through increased H2O2 production |
| Tomato | SlGRXS | Abiotic stress response | Gene editing (multiplex) | Developed mutants resistant to climate change |
| Tomato | SLARF4 | Salt tolerance | Knockout | Enhanced root development and chlorophyll maintenance |
| Tomato | SIHAK20 | Na+/K+ion regulation | Knockout | Increased sensitivity to salt stress |
| Tomato | HyPRP1 | Salt tolerance | Knockout | High salinity tolerance in mutants |
| Tomato | SIABIG1 | Salt resilience | Knockout | Developed salt-resilient tomato variety |
| Tomato | SP5G | Early flowering | Gene editing | Induced early flowering under changing climatic conditions |
| Tomato | SLCBF1 | Chilling stress | Knockout | Increased sensitivity to chilling stress |
| Tomato | SLUVR8 | UV-B stress tolerance | Knockout | Improved seedling development under UV-B stress |
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