*6.2. CRISPR*/*Cas9 for the Production of Climate Smart Crops*

The CRISPR/Cas9 technology has been extensively applied in major crop plants such as wheat, rice, maize, cotton, soybean, tomato, and potato to cope with various abiotic stressors. Development of climate smart abiotic stress-tolerant crops via the CRISPR/Cas9 tool has modernized plant breeding programs. Major events for crop improvement via CRISPR/Cas9 are described in Table 4.

For example, in wheat protoplast, two genes related to abiotic stress, *TaDREB3* and *TaDREB2*, have been studied using the CRISPR/Cas9 technique. With a T7 endonuclease assay, the expression of mutated genes has been confirmed in approximately 70% of transfected protoplasts. The mutated plants showed increased tolerance against drought as compared to wild cultivars [221]. Three rice genes named mitogen-activated protein kinase (*OsMPK2*), phytoene desaturase (*OsPDS*), and betaine aldehyde dehydrogenase (*OsBADH2*) have been edited using the CRISPR/Cas9 technique. For transformation of CRISPR/Cas9 machinery, particle bombardment and protoplast transformation methods were used and revealed that these genes are responsible for regulating many abiotic stressors [26].

To protect plants from abiotic stressors, plant annexins play a major role. The annexin *OsAnn3* gene in rice has been studied under cold stress and its function was determined in edited knockouts developed by the CRISPR/Cas9 system [222]. Similarly, the gene *SAPK2* was mutated to study the stress tolerance mechanism in rice. The results revealed that the expression level of *SAPK2* was enhanced under drought and salinity stress conditions [223]. Drought tolerance in transgenic maize was enhanced by the overexpression of *AGROS* genes and they are of great prominence for maize breeding. To identify new allelic variants, CRISPR/Cas9 was applied to mutate the *ARGOS8* gene [224]. Curtin and colleagues carried out CRISPR/Cas9-based knockout mutagenesis of two genes *Drb2a* and *Drb2b* and found that these genes regulate salt and drought tolerance in soybean [225]. In tomato, important signaling molecules, i.e., mitogen-activated protein kinases (*MAPKs*) that respond against drought stress by protecting the membrane of cells from oxidative destruction and regulating genes transcription to tackle drought stress. The association of the *SlMAPK3* gene in controlling the drought tolerance mechanism has been reported in tomato by creating knockout mutants of the *SlMAPK3* gene under drought stress through the CRISPR/Cas9 system [226].

Many important traits like stress tolerance and crop yield are controlled by multiple genes. Many studies have been carried out to locate the quantitative trait loci (QTLs) that are controlling important traits in crop improvement programs. For the development of better performing varieties, such QTLs have been transferred into the elite lines. But this introgression is laborious for closely associated QTLs and if non-target regions are introduced into best performing varieties, it may produce many deleterious effects. Conversely, CRISPR/Cas technology can be a fascinating approach to generate and examine targeted mutagenesis. Using a CRISPR/Cas9-mediated QTL editing approach, the functions of grain number QTLs (*Gn1a*) and grain size (*GS3*) in rice varieties were examined [227]. Hence, the above studies revealed that CRISPR/Cas9-based GE has massive potential for the development of climate-resilient crops.


**Table 4.** Summary of CRISPR/Cas9 applications in major crops for abiotic stress tolerance.

pseudogene (*ARGOS8*), Mitogen-activated protein kinase 3 (*SlMAPK3*), Mitogen-activated protein kinase 2 (*OsMPK2*).
