*4.4. CRISPR-Cas Mediated Genome Editing*

Tolerance relative to high temperatures is mostly regulated by many genes based on the degree of stress and the stress tolerance mechanism. It will be more difficult to target a tolerance mechanism that is controlled by multiple genes. Plant heat stress response is precisely regulated by a complex web of TFs from distinct families. These TFs improve plant heat stress tolerance by modulating the expression of several stress responsive genes, either individually or in conjunction with other regulatory factors. There are numerous successful genetic engineering applications inducing heat stress tolerance in plants using heat stress TFs and HSPs genes [165,166]. However, genetically modified crop plants are subject to stringent regulatory requirements, which may cause lab research to be delayed in reaching the market. As an alternative to traditional transgenic approaches, recently emerged CRISPR-Cas-mediated genome editing allows researchers to alter, modify or swap alleles, and insert or silence gene(s) in a predefined manner [167].

High temperatures alter the expression pattern of several plant genes either by upregulating or down-regulating them. Although our understanding of differentially expressed genes in response to drought and salt stress has expanded, relatively less focus has been made on studying heat stress associated genes in cotton. Studying the expression pattern of heat stress responsive genes in cotton under long-term heat stress clearly showed that expressions of *HS126*, *HS128*, *FPGS*, *TH1*, and *IAR3* genes increased under high temperature. In contrast, the expressions of *ABCC3*, *CIPK*, *CTL2*, *LSm8*, and *RPS14* genes were downregulated [153]. Therefore, targeted modulation of these up-regulated and down-regulated genes in cotton using the CRISPR-Cas system would be an exciting opportunity for combating the negative impact of heat stress. Moreover, multiple HSPs and TFs associated with heat stress sensitive genes have been proposed as potential candidates for improving plant heat tolerance [168]. Therefore, understanding the exact role of these genetic regulators paves the way for the development of enhanced heat tolerance, while maintaining overall plant resilience. In maize plants, peak photosynthesis has been observed between 20 and 32 ◦C, and the subsequent increase in temperature caused a decrease in net photosynthesis rate depending on plant growth stage [169]. CRISPR/Cas9 mediated disruption of the heat-stress sensitive albino-1 (*HSA1*) gene in rice exhibits higher heat sensitivity compared to wild plants [170]. The *slagamous-like 6* (*Slagl6*) gene was reported as a potential gene in the study of facultative parthenocarpy. Thus, researchers have successfully developed heat-tolerant parthenocarpic tomato fruit by mutating the *SlAGL6* gene using the state-ofthe-art CRISPR-Cas9 system [171]. In addition, thermosensitive male sterile maize lines have also been developed by mutating the *thermosensitive genic male-sterile 5* (*TMS5*) gene using CRISPR-Cas9 editing [172].

CRISPR-Cas9 has been modified and exploited for a range of new functions, including controlling gene regulation by activating and suppressing target gene expression using CRISPR activation and interference systems [173]. Positive gene regulators associated with HSPs and stress related TFs could be activated through the CRISPR activation system with high specificity. Moreover, negative regulators could be knocked out by using the CRISPR interference system. In one of study, the *BZR1* gene was up-regulated and repressed using CRISPR activator and interference systems. The results show that the overexpression of the *BZR1* gene enhances H2O2 production and recovery of thermo tolerance in rice, while plants with suppression of the gene show impaired production of H2O2 in apoplast and reduced heat tolerance [174]. Previously, the roles of *MAP3Ks* remained poorly understood in cotton. Recently, it has been reported that *MAP3K65* gene expression is induced by multiple signaling molecules, pathogen infection, and heat stress. This gene enhances susceptibility to pathogen infection and heat stress by negatively modulating growth and development related processes. Moreover, silencing *GhMAP3K65* enhanced resistance to pathogen infection and heat stress in cotton. Therefore, *GhMAP3K65* is a potential candidate gene to target with the CRISPR-Cas9 genome editing system in order to engineer heat tolerance in cotton [175]. In conclusion, maneuvering positive and negative regulators of heat stress signaling molecules in cotton could, thus, be exploited to develop new cotton cultivars tolerant to extreme temperatures.
