**1. Introduction**

By the end of the year 2050, the world population is anticipated to reach up to 10 billion [1]. In this situation, increasing food crop production by 60% over the coming decades is necessary to ensure global food security [1,2]. To sustainably increased food production, additional integration of all developed relevant techniques, such as genomics, genome editing (GE), artificial intelligence, and deep learning, will be necessary [3,4]. Crop modification methods have a long history and have been used ever since the first agricultural plants were domesticated. Since then, other new methods have been created and are being developed to boost crop production and economic value even more. Traditional crop breeding techniques in the 20th century either relied on naturally occurring mutations or on mutagenesis that was created artificially [5]. Genetic research has traditionally focused on the identification and assessment of spontaneous mutations. Scientists were reliant on each other and showed that radiation or chemical treatment could increase the rate of mutagenesis [6,7]. Later approaches, suchas radiation and chemical mutagenesis, altered the genome at random sites by inserting transposon motifs that may be induced in some

**Citation:** Yadav, R.K.; Tripathi, M.K.; Tiwari, S.; Tripathi, N.; Asati, R.; Chauhan, S.; Tiwari, P.N.; Payasi, D.K. Genome Editing and Improvement of Abiotic Stress Tolerance in Crop Plants. *Life* **2023**, *13*, 1456. https:// doi.org/10.3390/life13071456

Academic Editors: Balazs Barna, Hakim Manghwar and Wajid Zaman

Received: 30 May 2023 Revised: 25 June 2023 Accepted: 26 June 2023 Published: 27 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

animals. However, a fundamental disadvantage of conventional breeding methods is the length of time needed to breed new varieties of any crops with the required agronomic characteristics. The duration of the growing season and the maturity level of the plants (particularly long-period growers, such as trees), as well as various stages of crossing, selection, and testing during the breeding process, all have an impact on this [8]. The plant genome cannot be targeted using conventional techniques for chemical and physical mutagenesis or natural mutations. Using genetic engineering, better plants and animals may be developed more quickly [5].

The first genetically modified (GM) crops were released for sale in 1996 [9]. Generations of GM crops up to now have relied on the genome's random insertion of new DNA sequences. The possibility that the inserted gene may affect or impede the activity of other crucial nearby genes has been raised as a concern regarding this approach. In addition, public anxiety regarding GM crops is increased when talking about the introduction of 'alien' genes from distantly related organisms, which is thought to be 'unnatural' despite mounting evidence to the contrary [10,11].

The creation and use of DNA-based markers at the turn of the twenty-first century has made it possible to reduce significantly the time needed to generate new lines and varieties of agricultural crops [10–13]. All these factors have greatly helped the development of focused GE methods [14–17]. In yeast and mice, the first targeted genetic alterations were created in the 1970s and 1980s [6,8]. This gene targeting was based on the homologous recombination process, which was extremely accurate.

RNA interference (RNAi) was one of the first GE technologies [5,18,19]. Even though this technology has been successfully used in functional genomics and plant breeding [20–22], it has several drawbacks, including the unlimited insertion site of an RNAi construction into the genome and partial gene function suppression [5].

This is a marvelous time for genetics, due to advances in genetic analysis and genetic manipulation. Genome editing, the most recent crop-enhancement method, allows precise changes of the plant genome by deleting undesired genes or enabling genes to acquire new functions [23]. Numerous crops' genomes have been sequenced, and improvements in genome-editing techniques have made it possible to breed for desired features. To sustainably increase food production, additional integration of all developed relevant techniques, such as genomics, genome editing (GE), artificial intelligence, and deep learning, is necessary [24].

Advanced biotechnological methods are made possible by genome-editing tools, allowing for precise and effective targeted modification of an organism's genome. Several novel tools for genome or gene editing are available to enable researchers to modify genomic sequences precisely [25]. These techniques facilitate novel insights into the functional genomics of an organism and enable us to alter the regulation of gene expression patterns in a pre-determined region. Because of accurate DNA manipulation, genome-editing technologies, for instance, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated systems), TALENs (transcription activator-like effector nucleases), CRISPR/Cas12a (Cpf1, CRISPR from *Prevotella* and *Francisella*1), and Cas9-derived DNA base editors, provide unprecedented advancements in genome engineering. As a result, this technology is a powerful tool that can be employed to secure the global food supply [26].

Genome editing was first proposed by Capecchi [27] in the 1980s. This method allows for the removal, modification, or addition of genetic material at specified genomic locations. Even though current GE technologies are substantially more accurate than traditional mutagenesis [28,29], the biggest barrier here is still the legitimacy of GE crops. Assessing the biosafety of such crops is a unique difficulty because it is impossible to predict the effects of single base alterations following the application of ODM and BEs [30,31].

The primary elements that affect plant growth and reduce agricultural productivity are biotic stressors [32,33] such as disease and insect pests, along with abiotic stressors [13] including cold, drought, and saline–alkali stress (Figure 1). Many crop plants that can withstand abiotic stress have previously been created via traditional marker-assisted breeding. However, due to extensive screening [34,35] and backcrossing procedures, it takes this tactic about a decade to generate abiotic stress-resilient crops effectively [36]. Although genetically modified, stress-tolerant plants have disclosed encouraging results, several barriers still stand in the way of their widespread commercialization. In many ways, crops with genome editing differ from genetically engineered species [37]. Considering this, genome editing seems to be a sophisticated strategy to create crops that are resistant to different abiotic stress in the future, because it allows precise manipulation of different gene loci in comparably less time, which lowers the cost of crop-improvement programmes [38]. Gene-editing technology based on CRISPR/Cas might successfully target complex quantitative genes linked either directly or indirectly to abiotic stressors. The use of CRISPR-Castechnology has been linked in recent years to the establishment of disease resistance in plants by modifying gene regulation [39–42]. Currently, CRISPR/Cas-based genome editing has been efficaciously utilized to investigate tolerance against multiple abiotic stresses, including heat, drought, salt, and nutritional values in several critical agricultural plants [43,44]. In this review article, we summarize the most likely uses of the CRISPR/Cas9-mediated genome editing technique in crop plants for dealing with diverse abiotic stresses such as heat, drought, salinity, cold, herbicide etc., and we predict the tools for future advancements in the creation of crop varieties that can withstand stresses.

**Figure 1.** Applications of genome editing in crop improvement against abiotic stresses.

#### **2. Genome-Editing Strategy**

Genome editing is one of the most promising approaches to understand the genome and to improve crop plants. The fundamental mechanisms involved in genetic modification by programmable nucleases (NHEJ) are the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-stranded breaks (DSBs) in target DNA caused by restriction endonucleases (FokI and Cas), and repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining [45]. While the well-organized and error-prone NHEJ results in the deletion or insertion of nucleotides, the

less efficient and more accurate HDR results in the replacement of nucleotides. Genomeediting methods such as ZFN, TALEN, and CRISPR/Cas are being utilized to add the desired trait(s) and remove the undesirable ones. Numerous techniques are available for genome editing using either a site-specific recombinase (SSR) system or a site-specific nuclease (SSN) system. Both systems must be able to find a known sequence. The SSN system causes single or double strand DNA breaks and activates endogenous DNA repair systems. Depending on how the sites (loxP, FLP, etc.) are oriented, SSR technology, such as Cre/loxP- and Flp/FRT-mediated systems, can knockdown or knock in genes in the eukaryotic genome around the area of the target [46].

Plant genome-editing techniques have been classified into four major types based on onsite-specific endonucleases (Table 1). Those are ZFNs, meganucleases, TALENs, and CRISPR-Cas9 along with DSB-free genome editing, base editing, prime editing, and mobile CRISPR. These techniques are all discussed in detail below.

#### *2.1. Zinc-Finger Nucleases*

ZFNs are assemblages of DNA recognition modules based on zinc fingers and the DNA cleavage domain of the FokI restriction enzyme. With their use, the target genome can be altered to introduce a variety of genetic changes, such as deletions, insertions, inversions, translocations, and point mutations [47]. They have two domains, the first of which is a nuclease domain and the second of which is a DNA-binding domain. The DNA-binding domain's 3- to 6-zinc finger repeats may recognize nucleotide sequences that are 9 to 18 bases long. The second domain is made up of the restriction enzyme Flavobacterium okeanokoites I (FokI), which is necessary for DNA cleavage [48].This method involves three artificial restriction enzymes, specifically ZFN-1, ZFN-2, and ZFN-3 [49]. ZFN-1: At this point, ZFN is transferred to the plant genome devoid of taking a repair template. Once it arrives at the plant genome, it makes double-stranded breaks (DSB) to the host DNA leading to non-homologous end joining (NHEJ) of DNA [50], which either produces site-specific arbitrary mutations or a small deletion or insertion. ZFN-2: Distinct from ZFN-I, a homology-directed repair (HDR) alongside a short repair template is delivered to the crop genome next to the ZFN enzyme [51]. The template DNA is homologous to the target DNA, which attaches to a specific sequence causing a double-stranded rupture. The template commences repairing with an endogenous repair mechanism which is directed to site-specific point mutations throughout homologous recombination (HR). ZFN-3: As soon as the ZFN transcribing gene is transferred to the plant genome next to the large repair template, it is called ZFN3 [51,52].

ZFN has been effectively implemented in *Arabidopsis*, tobacco, soybean, and maize [53–56]. In one example of the use of ZFNs in crop breeding, the insertion of PAT gene cassettes disrupted the endogenous ZmIPK1 gene in maize, which altered the inositol phosphate profile of growing maize seeds and improved herbicide resistance [53].ZFNs can be created utilizing various protein-engineering techniques to target essentially any unique DNA stretch [57]. ZFNs with enhanced specificity and activity have been developed to produce knockouts, which disable the gene's function, as well as gain-of-function alterations [58].

#### *2.2. Meganucleases*

Longer DNA sequences (more than 12 bp) can be selectively detected and cut by meganucleases, which are endonucleases. This approach has been discovered in a wide variety of organisms, including archaebacteria, bacteria, algae, fungi, yeast, and many plant species. Meganucleases at the target region can sustain mild polymorphisms [59]. Meganucleases have been divided into five groups based on their sequence and structural features. These consist of His-Cys box, GIY-YIG, LAGLIDADG, PD-(D/E) XK, and HNH [60,61].Genome editing has mostly used members of the LAGLIDADG meganuclease (LMN) family. According to Silvaet al. [60], the name of this protein family is taken from the sequence of the main motif found in its structure. LMNs are typically expressed in the chloroplast and mitochondria of unicellular eukaryotes. The bulk of these endonucleases

are dimeric proteins that have two separate functions: they splice their own introns as RNA maturases and cleave exon sequences as specialized endonucleases [62]. I-SceI and I-CreI's genomes can be edited employing the rRNA gene of the mitochondrial DNA of *Saccharomyces cerevisiae*. The 21S contains the I-SceI gene's location. The chloroplast of *Chlamydomonas reinhardtii*, a unicellular alga, was found to contain I-CreI, which is found in the 23S rRNA gene. However, due to the difficulties in reengineering meganucleases to target specific DNA areas, their utility in genome editing is limited [63].

#### *2.3. Transcription Activator-like Effector Nucleases (TALENs)*

Restriction enzymes called TALENs, or transcription activator-like effector nucleases, are designed to cleave specific DNA sequences. TALENs are made up of a nuclease that can cleave DNA in cells and a TALE domain that is intended to mimic the natural transcription activator-like effector proteins. Currently, a huge number of researchers are studying transcription activator-like effector nucleases (TALENs), which are composed of a free designable DNA-binding domain and a nuclease [64], in a variety of organisms. TALENs have recently emerged as a cutting-edge method for genome editing in a variety of species and cell types. It was discovered that TALENs may alter the genome in a variety of plants, including *Arabidopsis*, Nicotiana, Brachypodium, barley, potatoes, tomatoes, sugarcane, flax, rapeseed, soybean, rice, maize, and wheat [65,66]. According to a report, rice was the first crop in which TALENs technology was employed for enhancement. According to Li et al. [67], the main pathogen of blight disease (*Xanthomonas oryzae*) significantly reduces global rice production each year. By disrupting the genes for fatty acid desaturase (FAD), soybeans with high oleic acid and low linoleic acid levels were produced, improving the shelf life and heat stability of soybean oil [68,69]. TALENs are naturally occurring type III effector proteins created by *Xanthomonas species* that change the host plant's gene expression. The TALENs proteins comprise a nuclear localization signal, a transcriptional activation domain, and a core DNA-binding domain [70]. The nuclear localization signal helps TALENs enter the nucleus, whilst the activation domain activates the transcriptional machinery to start expressing genes [71].

#### *2.4. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated Protein 9 (Cas9)*

Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) are short, repetitive genetic variations that are present in most bacterial and archaeal species. CRISPR/ Cas9 and its associated proteins produce a very strong defensive system that works as a safeguard for plants against foreign agents including bacteria, viruses, and other elements. The first application of CRISPR/Cas9 in an adaptive immune system was documented in a 2007 experiment [72]. The CRISPR/Cas9 gene-editing system has revolutionized research in animal and plant biology since its usage in genome editing was first demonstrated in mammalian cells in 2012 [73]. According to Rathore et al. [23] first-generation CRISPR/Cas9 genome editing involves simple manipulationand cloning techniques that can be applied to a variety of guide RNAs to edit different locations in the targeted organism's genome (Figure 2). With the use of CRISPR/Cas, crop species can be precisely edited, opening the door to the generation of favorable germplasm and new, more sustainable agricultural systems. The genetic modification of crops can now be targeted and precise due to recent developments in CRISPR/Cas9 technology, hastening the advancement of agriculture [42]. To date, only a few species have been studied using this methodology [74].The yield, quality, disease resistance, and climatic adaptability of monocots and dicots have all been improved by the CRISPR/Cas9 system [75]. The genomes of cereal crops including wheat, maize, rice, and cotton as well as fruits and vegetables such as tomatoes and potatoes have all been altered using the CRISPR/Cas9 technique [76,77].

**Figure 2.** Mechanism of genome editing using CRISPR/Cas9.

According to Makarova et al. [78], the CRISPR/Cas system can be divided into three types: type I, type II, and type III. Bacteria and archaea both have type I CRISPR/Cas mechanisms based on the exact signature of the Cas protein. The Cas3 protein's endonuclease activity is used to connect to the DNA sequence [78]. In bacteria, the type II CRISPR/Cas system has been developed. The four protein pairs Cas1, Cas2, Cas4/Csn2 proteins, coupled with Cas9, make up the simplest system. The type III CRISPR/Cas system hunts for DNA and RNA in archaea, as well as infrequently in bacteria. Cas6, Cas10, and repeat associated mysterious proteins (RAMP) are markers for its presence. Cas10 protein's processing of crRNA ultimately aims to cleave DNA [78]. The *Streptococcus pyogenes* (SpCas9)-derived type II CRISPR system mostly targets the negatively regulating genes [79].

The CRISPR/Cas technique is straightforward, stable, and enables effective change compared withthe first two generations of genome-editing systems. These traits allowed CRISPR/Cas to quickly replace the traditional genome-editing methods ZFN and TALEN. The techniquewas adapted from the bacterial defense mechanism. The CRISPR/Cas mechanism is used by a variety of bacterial and archaeal species to protect themselves against invading viruses [80]. Many studies are now being conducted to improve the CRISPR/Cas system and increase the tool's ability to target the genome. For instance, non-canonical NGA and NG PAM sites in plants may be found using xCas9, SpCas9-VRQR, and Cas9-NG variants [81,82]. SpCas9 orthologues have been recognized from *Streptococcus thermophiles* (St1Cas9), *Staphylococcus aureus* (SaCas9), *Streptococcus canis* (ScCas9), and *Brevibacillus laterosporus* (BlatCas9).They have been demonstrated to amend plant genomic loci with PAM sequences of NNGRRT, NNG, NNAG AAW, and NNNCND, respectively [83,84]. Additionally, the type V Cas12a and Cas12b extracted from different bacterialsystems have been demonstrated with AT-rich PAM specifications and employed in genome editing of selected plants [85,86].

The CRISPR/Cas9 gene-editing approach has so far been used on more than 20 crop species to increase yields and reduce biotic and abiotic stress [87]. Genome-editing techniques based on CRISPR/Cas9 have been utilized to enhance agricultural disease resistance and tolerance to severe abiotic environments including salinity and drought. Three rice genes involved in regulating responses to various abiotic stress stimuli, including phytoene desaturase (OsPDS), betaine aldehyde dehydrogenase (OsBADH2), and mitogen-activated protein kinase (OsMPK2), have undergone sequence-specific CRISPR/Cas9-mediated genomic modification. CRISPR/Cas9 technology was successfully used by Shan et al. [88] to insert the TaMLO gene (mildew resistance locus O) into wheat protoplasts. It was also discovered that *Blumeria graminis* f. sp. Tritici, the agent of powdery mildew illness, is resistant to the CRISPR TaMLO knockdown (Btg). Wheat ethylene responsive factor3 (TaERF3) and wheat dehydration response element binding protein 2 (TaDREB2) are two abiotic stress-related genes that were targeted by the CRISPR/Cas9 genome-editing technology in wheat protoplasts, according to Kim et al. [89]. The CRISPR/Cas9 technology can be used in conjunction with current and upcoming breeding techniques such as speed breeding and omics-assisted breeding to boost agricultural production and ensure food security (Table 2).

**Table 1.** Comparison of different types of plant genome-editing techniques.


**Table 2.** List of reported targeted gene(s) via ZFNs, TALEN, and MNs gene-editing tool technologies in different plant species to develop resistant/tolerant genotypes.



**Table 2.** *Cont.*

## *2.5. DSB-Free Genome Editing*

A sole histidine residue at site 840 of the HNH domain of SpCas9 cuts the PAM strand, while the aspartate at site 10 in the RuvC domain cuts the opposite strand3. Mutating both amino acids to alanines (D10A and H840A) resulted in nuclease-dead Cas9 (dCas9). dCas9 still identifies its target site and frees up the DNA in an R-loop without including DSBs. The binding of dCas9 to its solitary target site can work as a repressor of transcription and is called CRISPR interference (CRISPRi). Alternately, dCas9 can be utilized as a tool for localization of DNA effector proteins to the genome. Examples of this approach are CRISPR– DNMT3 fusion proteins and CRISPR activators (CRISPRa) for targeted methylation. DNAalteration enzymes are combined with dCas9 to induce genetic variants for overcoming the limitations linked with DSB initiation in genome engineering [117].

#### *2.6. Base Editing*

The first base editor combines dCas9 to the cytidine deaminase apolipoprotein B mRNA editing catalytic polypeptide-like (rAPOBEC1), which catalyzes the alteration from cytidine to uracil. The cell mends this uracil into thymidine, resultingin an assembly (BE1) replacing a C•G by a T•A base pair, entitled a cytosine base editor (CBE) [118]. First-generation CBEs were suppressed by uracil glycosylation. So, second-generation base editors (BE2) were invented by combining an uracil glycosylase inhibitor (UGI) with the dCas9–rAPOBEC1 combination [119].For increasing editing efficiency, dCas9 can be changed into a nickase SpCas9-D10A (BE3). The strand not altered by rAPOBEC1 is cleaved. The cell identifies this nick and starts DNA repair to solve the damage. The strand withthe base modification is used as a template for repairing the nick to yield stable integration. The BE3 architecture was furthermore ameliorated by combining an additional UGI in fusion with linker optimization to result in a fourth-generation cytosine base editor (BE4). BE4s have improved editing efficiency by approximately50%, with two-fold decline of unintended byproduct formation such as point mutations and indels [118]. Subsequent ancestral reconstitution and codon optimization led to a CBE architecture that enables the

most powerful base editing in organoids, 2D cell lines, and in vivo by improving nuclear localization and expression of the proteins [120].

#### *2.7. Prime Editing*

The logic behind prime editing is to escort exogenous DNA with the modification of interest close to the Cas9 binding site. Areverse transcription (RT) domain obtained from the Moloney murine leukaemia virus was combinedwith nickase SpCas9- H840Atodevelopthe first generation of prime editors (PE1). The RT domain changes RNA into DNA tofind its template in the 3 extension of the specially designed sgRNA, entitledthe primeediting guide RNA (pegRNA).Itguides the Cas9 in PE1 to the target site. After targetrecogination, the PAM-consistingstrand is nicked by the active HNH domain of Cas9-H840A. Then, the pegRNA extension combineswiththe nicked strand of the primer-binding site (PBS).Then, the RT domain of PE1 uses the restpegRNA(RT template) to synthesize a 3- -DNA flap containingthe edit of interest. This DNAflap is solved by cellular DNA repair procedure combining the edit of interest [121]. Theprime editing requires optimizing PE3guides andpegRNA, limiting its implementationin organoids. Threemodifications have been made forovercoming this issue. First, the utilizationof two pegRNAs in trans alongwith overlayingRT domains enhancesprime-editing competencein plants [121]. Second, engineered pegRNAs can have tmpknot or evopreqdomains combinedatthe 3 end. These domains enhancethe stability of the pegRNA [122]. Finally, including the N394Kand R221K amino acid alterationincreases the nuclease workof SpCas9, resulting in a more efficient PE2Max [123].
