*2.8. Mobile CRISPR*

A breakthrough in the CRISPR tool, "genetic scissors" was announced by scientists of the Max Planck Institute of Molecular Plant Physiology to edit plant genomes. The discovery could speed up and simplify development of novel and genetically stable crop varieties by fusing grafting with a 'mobile' CRISPR tool. The drawing of the CRISPR/Cas9 gene scissors is transferred as RNA from the rootstock of a genetically modified plant to the grafted shoot of a normal plant. The gene scissors protein is made with the aid of the RNA. This gene scissor protein edits specific genes in flowers. Plants carry the desired gene modification in the next generation. A normal shoot is grafted onto roots containing a mobile CRISPR/Cas9, which allows the genetic scissor to move from the root into the shoot. It edits the plant DNA without leaving a trace of itself in the subsequent generations of plants. This ground-breaking turn can save cost and time and evade current limitations of plant breeding.

#### **3. Genome Editing Related to Abiotic Stresses**

Abiotic stresses that impact plant growth and development, such as salt, drought, extremely high temperatures, cold, and heavy metals, can reduce agricultural production by approximately 50% [124].Numerous biochemical, morphological, and physiological factors important for plant development are influenced by stress. Stresses from the environment can modify how plants behave as they develop. Most changes in plant growth and development caused by different abiotic stresses are associated with poorer yields [13]. By 2050, the rapid growth in the human population is predicted to reach 9.7 billion. The global temperature is also set to increase significantly. As plant scientists, it is hard for us to manage the food requirements of the increasing population. However, we own the capability to develop climate-flexible crop varieties that can flourish under such challenging circumstances. These varieties must be maintained in ruthless climatic conditions such as heat, drought, heavy metals, cold, or flood stresses. This requires a continuous search for newer and diverse germplasm [125,126], which was traditionally performed either entirely through development of natural variations [127,128] or by selective breeding [129,130]. Another possibility is the construction of mutant populations that are evaluated to hunt for new resources among variations that might be novel valuable mutations that in turn are included in breeding programmes. Modern genome-editing system tools such as CRISPR

facilitate the user to commence desirable genomic modifications accurately, illustrating great promise as a tool for producing novel climate-resistant plants [131]. In over 20 agronomically important crops, CRISPR/Cas mediated gene editing is widely utilized and accepted for crop improvement against different abiotic stresses [79].

Ordinarily, plants are equipped with numerous defense schemes against abiotic stresses. Among numerous defense mechanisms of abiotic stresses, the five broad-spectrum protections are regulated utilized in a complicated managing network consisting of numerous mediators and gene regulatory constituents in response to abiotic stresses [132]. During the procedure, stress hormones, particularly nitrogen oxides (NO), abscisic acid (ABA), polyamines (PAs), calcium ions (Ca2+), hydrogen sulfide (H2S), reactive oxygen species (ROS), and phytochrome B (PHYB), interact with others, either synergistically or antagonistically. The transcription factors (TFs) could alter the expression of genes and enzyme activity in a regulatory way, triggering a suitable reaction. The regulatory constituents open a lot of potential for developing multiple stress tolerance/resistance. Five main plant defenses to abiotic stresses are ROS scavengers, molecular chaperones, cuticle as the outer shield, oxylipin precursors, and osmoprotectants, along with unsaturated fatty acids, and compatible solutes [132].

#### *3.1. Drought Stress*

Drought is becoming a challenge to sustainable agriculture due to the consequences of climate change, including erratic rainfall patterns and rising temperatures in many regions of the world. The greatest danger to global food security is drought stress, which is the primary factor in the catastrophic loss of agricultural production and productivity [133]. Drought alone can reduce yield by 50–70% in different crops [134]. For example, 40% yield losses due to drought stress have been reported in maize [35,135], 50% in rice [136], 21% in wheat [126,135], 27–40% in chickpea [125,137], 68% in cowpea [138] and 42% in soybean [34,139]. After the discovery of genome editing, efforts are being planned to alter the genes involved in pathways enabling drought tolerance, in order to increase farmers' acceptance of crops using these technologies. In recent years, in-depth research has helped to adapt and overcome drought stress using CRISPR-Cas9 technology (Table 3).

In many crop plants, H2O2 and abscisic acid (ABA) are frequently produced in situations of salinity or drought stress. The discovery was reported of ABA-induced transcription repressors (AITRs) as a novel transcription factor family that plays a significant role as feedback regulators of ABA signaling. Alternation in the expression of AITR genes resulted in abiotic stress tolerance, including drought and salinity in *Arabidopsis* [140,141]. A CRISPR/Cas9-induced mutation in the *Arabidopsis* OST2 structural gene exhibited drought resistance [142]. Another study found that knockout of Arabidopsis plants' genemiR169athrough CRISPR/Cas9 led to significantly improved drought tolerance [143]. Similarly, Arabidopsis' drought tolerance increased after the vacuolar H+-pyrophosphate (AVP1) regulating gene was expressed using CRISPR/Cas9 [144]. Similar results were shown when the abscisic acid-responsive element binding gene (AREB1) was activated in Arabidopsis through CRISPR/Cas9a [145]. Recently, drought tolerance in *Arabidopsis thaliana* was demonstrated via the CRISPR/Cas9 gene silencing of the trehalose (TRE1) gene [146].

Numerous studies have documented how CRISPR confers drought resistance in many plants. For instance, it has been demonstrated that increasing rice's ability to withstand drought can be attained by reducing the expression of the regulatory genes DERF1, PMS3, MSH1, MYB5, and SPP [147]. In rice plants, drought stress tolerance increased after OsERA1 was modified using CRISPR/Cas9 [148]. CRISPR/Cas9 has been employed to improve drought resistance in rice by knocking out the SRL1, SRL2, and ERA1 genes [148,149]. A CRISPR/Cas9-created ospyl9 mutant might increase rice yield and drought tolerance [150]. Indica mega rice cultivar MTU1010 with broader leaves, a decreased stomatal density, and improved leaf water retention under drought stress was developed using CRISPR/Cas9 to modify the *OsDST* gene [151]. The *OsOREB1*, *OsRab21*, *OsRab16b*, *OsLEA3*, *OsbZIP23*, *OsSLAC1*, and *OsSLAC7* genes, which act downstream of SAPK2, were modulated in expression in the loss-of-function sapk2 mutant of rice plants developed using CRISPR/Cas, increasing their tolerance to drought stress [131].

Two genes, *RVE7* and *4CL*, have been found to be associated with drought tolerance in chickpeas. The first report of CRISPR/Cas9-mediatedediting of the chickpea protoplast was made by Badhan et al. [152]. They described knockouts of the genes *4CL* and *RVE7*, which are linked to pathways for drought tolerance. That study established a framework for potential future chickpea-genome-editing approaches [153]. Another gene, namely *ARGOS8*, responding to drought stress has been altered through genome editing. The expression of the *ARGOS8* gene increased as a result of negative regulators of ethylene signaling pathways, providing drought tolerance [154,155]. To increase the production of maize under drought stress under field conditions, the GOS2 promoter region was replaced with an *ARGOS8* promoter sequence using the CRISPR/Cas system [156].

CRISPR/Cas9 altered the *GID1* gene in tomato plants, which exhibit high leaf water content under drought conditions [157]. Additionally, *SlLBD40* gene mutation caused by CRISPR/Cas9 significantly improved drought tolerance in tomato [158]. Furthermore, use of the CRISPR/Cas technique to alter mitogen-activated protein kinases (MAPKs) revealed SlMAPK3 to be a drought stress modulator [159]. Knockout of the *SINPR1* gene resulted in increased drought tolerance and down-regulation of drought-related genes [160].

Drought resistance of wheat was improved by CRISPR/Cas editing of wheat *TaDREB2* and *TaERF3* [89]. In wheat, a multiplex CRISPR/Cas9 assay was used to alter the *SAL1* gene, a negative regulator of drought tolerance, to increase drought tolerance at the seedling stage [161]. CRISPR/Cas genome editing of the HB12 gene can increase cotton's resistance to drought [162]. CRISPR/Cas9 was used to modify the *BnaA6.RGA* gene in oil seed crops, which significantly improved rapeseed's ability to withstand drought [163].
