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/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

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, 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 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.

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

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).