CRISPR/Cas9-Based Genome Editing on Abiotic Stress Tolerance: Comparison
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Please note this is a comparison between Version 2 by Jason Zhu and Version 1 by Ji-Hoon Lee.
Plants are subjected to various environmental stresses that negatively impact growth and development and limit crop productivity. Therefore, in order to meet the requirements of the growing world population and food security, it is essential to develop cultivars resistant to abiotic stresses. In recent years, with the availability of genetic databases and the advancement in genome editing techniques, it is feasible to edit target genes with precision and create new opportunities for crop improvement that conventional breeding methods could not achieve. The genome-editing method using CRISPR-Cas systems is very powerful and confers exceptional versatility to develop improved cultivars at abiotic stresses. These efficient gene editing techniques facilitate the cultivation of superior-performing genotypes in challenging environmental conditions without compromising yield.
  • abiotic stress
  • CRISPR/Cas9
  • genome editing
  • genome databases

1. Introduction

Due to abiotic stresses, plant growth and development are affected, which can cause crop yield reduction at approximately 50% [113][1]. Though productivity increases to a large extent by traditional breeding, it may cause a loss of genetic variety and fitness. In addition to the development time period, it relies on natural allelic variants, which makes it challenging to develop the desired characteristic and to ensure the sustainability of production. Genome editing must include precise modifications at specific sites to perform desired changes to the DNA sequence [20,21,30][2][3][4]. Therefore, genome-editing techniques employing sequence-specific nucleases (SSNs) have become popular in plant research to develop improved cultivars in terms of yield, nutrition content, and resistance to environmental stresses. The SSNs introduce DNA DSBs at a target site, stimulating the cellular DNA repair and resulting in genome alterations, including targeted mutagenesis, gene insertion, and gene replacement [114][5]. In recent years, three types of genome-editing techniques have been widely used, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeat CRISPR/Cas protein systems. Due to complex procedures and high failure rates, ZFN and TALEN have not been utilized extensively, whereas CRISPR/Cas was successfully used in various crop improvement programs. CRISPR/Cas9-based genome editing is accurate because it targets specific sites of particular genes involved in stress response pathways and modifies genes to develop plants’ ability to withstand environmental stress [30,115][4][6]. Additionally, the CRISPR/Cas system has been used to introduce critical agricultural traits, including plant resistance against abiotic and biotic stress, and other agronomically important traits (increased grain size and grain weight) into many economically important crops, such as O. sativa, T. aestivum, Z. mays, L. esculentum, S. tuberosum, N. tabacum, Gossypium spp., G. max, Brassica sp., S. italica, and Saccharum spp. [5,30,31,32,33,34,37,39,40][4][7][8][9][10][11][12][13][14].

2. Improvement in Drought Stress Tolerance using CRISPR/Cas System

Drought stress can reduce crop yields by 50–70% in different crops due to significant reductions in plant growth and development. For example, a 27–40% yield reduction has been observed in C. arietinum, 42% in G. max, 50% in O. sativa, 21% in T. aestivum, 68% in V. unguiculata, and 40% in Z. mays [5][7]. Plants experience morphological, physiological, biochemical, and molecular changes in response to drought stress. CRISPR/Cas technique was successfully applied to enhance the drought resistance of rice crops by modifying the expression of drought and other stress-related genes [116][15]. The potential of CRISPR/Cas gene editing has been documented in various crop species against drought stress (Table 3). Researchers aimed to enhance plants’ ability to withstand drought stress and reduce crop losses by altering drought-related genes. A truncated version of gRNAs (<20 nucleotides) with target sequences in plant cells was used to improve the specificity of CRISPR/Cas9 and eventually generate altered alleles for OST2 (Open Stomata 2). The novel mutant alleles for OST2 exhibited drought tolerance by altered stomatal closing in response to environmental stress in A. thaliana [117][16]. Similarly, the remodeled CRISPR/Cas9 activation system activates vacuolar H+-pyro phosphatase AVP1, leading to an increase in single-leaf area, an increase in leaf numbers, and an enhancement of stress tolerance to drought [3][17]. Improved drought resistance was found in homozygous CRISPR/Cas9-edited MIR169a T3 plants using a combinatory dual-sgRNA/Cas9 vector containing deleted miRNA gene regions (MIR169a and MIR827a) [118][18]. Histone acetyltransferase (HAT) modifies chromatin histone, exposing DNA to the transcriptional machinery and regulating gene expression. Stable transgenic plants expressing chimeric dCas9HAT in A. thaliana showed higher chlorophyll content, faster stomatal aperture, and an improved survival rate under drought-stress conditions [119][19]. Trehalase (TRE1) gene silencing through the use of the CRISPR/Cas9 system developed drought tolerance in A. thaliana [120][20]. Transcriptome analysis using microarray technology is the best technique that has proven helpful in discovering many stress-inducible genes/stress-inducible transcription factors including the DRE-binding protein (DREB) members, ethylene-responsible element binding factor (ERF), zinc-finger, WRKY, MYB, basic helix-loop-helix (bHLH), basic-domain leucine zipper (bZIP), NAC (NAM, ATAF1, and CUC2), and homeodomain transcription factor families [4][21]. Overexpressing AtNAC07, AtNAC019, and AtNAC055 can enhance tolerance to drought in A. thaliana [121][22]. Dehydration-responsive element binding (DREB) proteins are one of the most prominent transcription factors and have a significant role in signaling networks regulating various plant development processes and stress responses. The overexpression of DREB1A/CBF3 (C-repeat binding factor) under the stress-inducible RD29A promoter improved drought tolerance in transgenic T. aestivum [122][23]. Drought tolerance in T. aestivum was enhanced by altering Dehydration-responsive element binding 2 (TaDREB2) and Ethylene Responsive Factor 3 (TaERF3) using the CRISPR/Cas system [123][24].
Abscisic acid (ABA) plays a vital role in drought tolerance by regulating the expression of many drought-related genes. ABA regulates the expression of genes through ABA-responsive element (ABRE) binding protein/ABRE binding factor (AREB/ABF). Drought stress tolerance has been demonstrated by over-expression of AREB1, as compared to the AREB1 knockout mutant [124][25]. In A. thaliana, ABF1, ABF3, AREB1/ABF2, and AREB2/ABF4 are expressed in response to ABA and drought stress in vegetative tissues, whereas ABI5, AREB3, DPBF2, and EEL are expressed during seed maturation [124,125,126][25][26][27]. Abscisic acid (ABA) signaling is regulated by ABA-induced transcription repressors (AITRs). The CRISPR/Cas9 system was used in soybean (Glycine max) to target the six GmAITR genes and generated Cas9-free gmaitr36 double and gmaitr23456 quintuple mutants, enhancing salinity tolerance [41][28]. Similarly, the Dehydration-responsive element [DREB1]/CBF is responsible for the ABA-independent induction of several genes in response to osmotic and cold stress, for example, RD29A/COR78/LTI78 gene in A. thaliana. The lateral organ boundaries domain (LBD) genes play essential roles in lateral organ development. CRISPR/Cas9 knockout of SlLBD40 improved drought tolerance in L. esculentum compared with overexpressing transgenic and wild-type plants [127][29]. Mitogen-activated protein kinases (MAPKs) are important signaling molecules that respond to drought stress. Similarly, the CRISPR-Cas knockout mutant for the SlMAPK3 gene down-regulated the expressions of drought stress-responsive genes: SlLOX, SlGST, and SlDREB [128,129][30][31]. The CRISPR-Cas9 mediated dst∆184–305 mutation in the DST (drought and salt tolerance) gene of O. indica cv. MTU1010 produced mutants having broader leaves and reduced stomatal density, resulting in improved leaf water retention under drought stress [130][32]. The SNF1-related protein kinase 2 (SnRK2) is the primary regulator of hyper-osmotic stress signaling and abscisic acid (ABA)-dependent plant development. A knockout mutant of the SAPK2 gene improved drought tolerance in O. sativa by affecting ABA signaling [131][33]. The CRISPR/Cas9-mediated knockout of SRL1 and SRL2 (Semi-rolled leaf 1, 2) and ERA1 (Enhanced Response to ABA1) genes improved drought tolerance in O. sativa. OSERA1 mutant lines display similar leaf growth as wild-type plants but enhanced primary root growth [132][34]. The SRL1 and SRL12 knockout mutants had fewer stomata, a slower rate of transpiration, less chlorophyll, vascular bundles, and rolled leaves than the wild type [133][35]. Plant ITPKs (Inositol trisphosphate 5/6 kinases) participate in abiotic stress signaling, and the itpk1 mutant created using programmable nuclease Cas9 displayed higher tolerance to salinity stress than deletion mutants in H. vulgare [134][36]. In B. napus, the bnaa6.rga mutant generated through CRISPR/Cas9 showed enhanced tolerance to drought stress by promoting stomatal closure through increased ABA sensitivity [40][14].
ARGOS is a negative regulator of the ethylene response, and CRISPR/Cas9-mediated editing of the ethylene response factor ARGOS8 improved drought tolerance in Z. mays [135][37]. WRKY are plant-specific transcription factors that play essential roles in abiotic stress response. Several WRKY transcription factors were identified in plant species, including A. thaliana, O. sativa, G. max, T. aestivum, and H. vulgare [136,137,138][38][39][40]. Overexpression of ZmWRKY40 promoted root growth and reduced the water loss rates in transgenic A. thaliana under drought stress [137][39]. Overexpression of the T. aestivum TaWRKY33 enhanced the drought and heat tolerance in transgenic A. thaliana [136][38]. OsWRKY5 is expressed in developing leaves at the seedling and heading stages of O. sativa. It is the negative regulator of drought, and its expression was reduced under drought stress and by treatment with NaCl, mannitol, and abscisic acid (ABA) [138][40]. These studies indicated the efficiency of the CRISPR/Cas system in developing drought-tolerant cultivars by knocking out or overexpressing target genes through precise genome editing.

3. Improvement in Salinity Stress Tolerance Using CRISPR/Cas System

Genome editing and genetic engineering tools have been utilized to target genes involved in ion transport for regulating osmotic adjustment under salt stress. Soil salinity is a critical abiotic stress affecting crop productivity worldwide. Plant salt tolerance is the ultimate manifestation of several physiologic processes, including Na+ uptake and exclusion, ionic balance (especially Na+/K+ ratio), and distribution [6][41]. Salt Overly Sensitive 1 (SOS1) is an extensively characterized Na+ efflux transporter in G. max, A. thaliana, and T. aestivum. The gmsos1 mutants were generated using the CRISPR-Cas9 system in G. max, and the resulting mutant displays a significant accumulation of Na+ in the roots and increased salt sensitivity [139][42]. In A. thaliana, the SOS signal transduction pathway (including SOS1, SOS2, and SOS3 genes) is essential for ion homeostasis and salt tolerance. The SOS1 gene isolated from durum wheat (T. durum) conferred salinity tolerance to the sos1 mutant of A. thaliana [140][43]. Similarly, the CRISPR/Cas9 knockout of the AITR family genes (AITR3 and AITR4) in A. thaliana enhanced tolerance to drought and salinity stress without fitness costs [7][44]. A gene cluster containing (T5G46490, AT5G46500, AT5G46520) and (NLRs; AT5G46510) is involved in osmotic stress tolerance. CRISPR/Cas9-mediated mutagenesis generated single and double knockout lines for ACQOS alleles in A. thaliana. A. thaliana plants containing complete deletions or pseudogenization-induced polymorphisms in ACQOS and AT5G46510 show considerable tolerance to salt stress, suggesting the role of ACQOS in salt stress tolerance [141][45].
Nitric oxide (NO) plays a vital role in cytoprotection by regulating the level of ROS and inducing transcriptional changes, leading to the modulation of protein function [142][46]. Reactive oxygen species (ROS) are highly reactive molecules typically produced in response to environmental stress, such as salinity and drought. Histone acetyltransferase TaHAG1 is a vital regulator to strengthen the salt tolerance of T. aestivum. TaHAG1 contributed to salt tolerance by modulating ROS production and signal specificity. CRISPR-mediated mutagenesis of TaHAG1 validated the role of TaHAG1 in salt tolerance in T. aestivum [143][47]. Salt stress increases ROS production and is responsible for oxidative damage, membrane injury, lipid peroxidation (malondialdehyde), and ultimately cell death. CRISPR/Cas9-mediated mutagenesis of the osbhlh024 gene negatively regulates the functions of Na+ and K+ transporter genes, suppressing the higher accumulation of MDA and H2O2, leading to salt tolerance in O. sativa [144][48].
Several quantitative trait loci (QTLs) and genes associated with regulating salt stress tolerance have been identified in O. sativa, including the NHX family (OsNHX1, OsNHX2, OsNHX3) [145[49][50][51],146,147], HKT family (OsHKT1, OsHKT2, OsHKT7) [148,149[52][53][54][55],150,151], DCA1 [152][56], DST1 [130[32][57],153], OsKAT1 [154][58], OsBADH1 [155][59], OsNAC5 [156][60], OsbZIP71 [157][61], SKC1, OsHAL3, P5CS, SNAC2, OsNAP, OsRRY [158[62][63],159], and OsSALP1 [113,160][1][64]. CRISPR/Cas9-mediated knockout of several salt stress genes significantly improved salinity tolerance in various crops. CRISPR/Cas9 and third-generation hybrid rice system approaches were employed to generate the OsRR22 mutant, which exhibited enhanced salinity tolerance without any morphological and physiological changes relative to the wild-type [159][63]. A receptor-like kinase gene OSBBS1/OsRLCK109 played vital roles in leaf senescence and salt stress response [161][65]. CRISPR/Cas9-mediated editing of the SAPK1 and SAPK2 genes showed resistance to salt stress in O. sativa [131][33]. The mutant alleles of DST (drought and salt tolerance) generated using the CRISPR/Cas9 method showed reduced stomatal density by downregulating stomatal developmental genes (SPCH1, MUTE, ICE1), resulting in a high level of salt tolerance in the seedling stage of O. sativa [130][32]. Argonaute (AGO) proteins primarily function in gene silencing by forming RNA-induced silencing complexes. CRISPR/Cas9-mediated AGO2-knockout mutant lines showed few morphological changes compared to wild-type rice. The overexpression of AGO2 under the control of the cauliflower mosaic virus 35S led to a simultaneous increase in salt tolerance and grain length [162][66]. Transcription factors such as AP2/ERF, NAC (NAM, ATAF1/2, CUC2), and WRKY families induce stress-responsive gene expression in response to environmental signals. APETALA2/ethylene response factor (AP2/ERF) plays crucial roles in transcriptional regulation and defense response against biotic and abiotic stress. Editing of the OsRAV2 (AP2/ERF domain-containing RAV) gene using CRISPR/Cas9 showed tolerance to salt stress [163][67]. DOF transcription factor (DNA-binding with one finger) regulates the elongation of the primary root positively by controlling cell proliferation in the root meristem by restricting ethylene biosynthesis. O. sativa mutant osdof15 showed reduced cell proliferation and primary root elongation in the root meristem [164][68]. A knockout mutant (ospqt3) with CRISPR-Cas9 technology displayed greater resistance to oxidative and salt stress with high expression of OsGPX1, OsAPX1, and OsSOD1 [165][69]. Similarly, CRISPR/Cas9 knockout of OsmiR535 demonstrated salinity tolerance in O. sativa against NaCl, ABA, dehydration, and PEG stresses [166][70]. OsNAC45 plays a vital role in ABA signal responses, and overexpression of NAC45 enhances salt tolerance in O. sativa. OsNAC45 may regulate the expression of seven genes namely CYP89G1, DREB1F, EREBP2, ERF104, PM1, SAMDC2, and SIK1 [167][71]. Targeted mutagenesis of the OsOTS1 gene using the CRISPR/Cas9 system in the O. sativa cv. Kitaake enhanced sensitivity to salt with reduced root and shoot biomass, indicating that OsOTS1 has a major role in salt stress tolerance [168][72].
Hormones like Gibberellic and Absiscic acid signaling pathways significantly affect salt stress. OsPIL14-SLR1 (Phytochrome Interacting Factor-Like14–DELLA protein, SLENDER RICE1) controls seedling growth in response to salt stress. CRISPR/Cas9 mediated ospil14 mutants produce normal mesocotyls and longer roots than wild-type plants [169][73]. ZmWRKY114 is a negative regulator of salt-stress responses, and overexpressed WRKY114 exhibited enhanced salt-stress sensitivity and reduced ABA sensitivity [170][74]. Salinity stress tolerance was identified in several stress-related genes like HyPRP1 (Hybrid proline-rich protein 1), HKT1, HKT1 (High-affinity potassium transporter1;2), RAD51/54 (DNA repair and recombination protein 51/54) and PR-1 (Pathogenesis-related protein 1) [37,151][12][55]. HyPRP1 is a negative regulator of salt stress responses, and CRISPR-Cas9 mediated genome editing of HyPRP1 in L. esculentum resulted in the elimination of the functional domain of proline-rich protein. Plants carrying such variants, PR1v1 lacking proline-rich domain, PR2v2 and PR2v3 lacking eight cysteine motifs, showed improved germination compared to wild type under osmosis stress [37][12]. A significant improvement in Homology-directed repair (HDR) using CRISPR/LbCpf1-geminiviral multi-replicons was reported to target marker-free salt-tolerant HKT1, HKT2 alleles in L. esculentum [151][55]. Self-pollinated offspring plants carrying the HKT1, and HKT2 allele showed stable inheritance and germination tolerance under salt stress conditions (100 mm NaCl concentration). In Z. mays, Na+ Content1 (ZmNC1) encodes an HKT-type transporter ZmHKT1, preferentially expressed in root stele. CRISPR/Cas9 knockout lines of ZmHKT1 increase Na+ concentration in xylem sap and cause increased root-to-shoot Na+ delivery, indicating that ZmHKT1 promotes leaf Na+ exclusion and salt tolerance by withdrawing Na+ from the xylem sap [150][54]. Mutations in genes OsRR9 and OsRR10 generated using the CRISPR/Cas9 system enhanced salinity tolerance but reduced panicle and spikelet numbers per panicle in O. sativa [171][75]. CRISPR/Cas9 mediated mutagenesis of the ARF (Auxin Response Factors) gene generates a slarf4 mutant that displayed salinity and drought tolerance in L. esculentum by stimulating root development and stomatal function [172][76]. These studies demonstrate the potential role of CRISPR/Cas mutagenesis in knocking out genes responsible for salinity tolerance in plants.

4. Improvement in Heat Stress Tolerance Using CRISPR/Cas System

Heat stress is the third most crucial abiotic factor that adversely affects the yield and quality of plants during entire growth stages, from germination to harvesting. Plants respond to heat stress in various ways, including alterations in enzymes that generate reactive oxygen species (ROS), heat shock proteins (HSPs), and genes encoding scavenger proteins [8][77]. The advancement of structural and functional genomics technologies in plants has led to the identification and characterization of various temperature-stress-related genes to enhance plant ability to withstand heat [9][78]. The heat-shock-induced CRISPR/Cas9-mediated genome editing efficiently produces heritable targeted mutations. In O. sativa, a heat-shock-inducible CRISPR/Cas9 system was employed to generate targeted and heritable mutations [173][79]. Similarly, CRISPR/Cas9-based genome editing targeted the heat-sensitive gene, Slagamous-Like 6 (SIAGL6), resulting in increased fruit setting under heat stress conditions in L. esculentum [174][80]. Calcium-dependent protein kinase 28 (cpk28) mutant was generated using CRISPR/Cas9 mediated editing and displayed thermotolerance in L. esculentum [175][81]. Brassinazole Resistant 1 (BZR1) is involved in thermo-tolerance by regulating the Feronia (FER) homologs. CRISPR/Cas9-based bzr1 mutant reduced apoplastic reactive oxygen species (H2O2) production and enhanced heat tolerance L. esculentum [176][82]. Photosynthetic apparatus is highly susceptible to thermal damage. Heat-sensitive albino1 (hsa1) mutant harbors a recessive mutation in a gene encoding fructokinase-like protein2 (FLN2), resulting in a severe albino phenotype with defects in early chloroplast development. In O. sativa, hsa1 mutants showed increased sensitivity to heat stress but had a faster greening phenotype than wild-type plants [177][83]. Knockout of the ZmTMS5 gene of Z. mays using the CRISPR/Cas9 system generated homozygous T1 tms5 thermosensitive male-sterile plants that are male-sterile at 32 °C but are male-fertile at 24 °C [178][84]. NCED4 (9-cis-Epoxycarotenoid Dioxygenase4) is a key regulatory enzyme in the biosynthesis of abscisic acid (ABA). Similarly, stable homozygous NCED4 mutants generated using CRISPR/Cas9 were capable of germinating seeds at a higher temperature (>70% germination at 37°) in Lettuce (Lactuca sativa) [179][85]. Another transcription factor, OsNAC006, is regulated by temperature stress, hormones (abscisic acid, indole-3-acetic acid, and gibberellin), NaCl, polyethylene glycol, and reactive oxygen species. Furthermore, CRISPR-Cas9 mediated knockout of OsNAC006 causes drought and heat sensitivity in O. sativa [180][86]. These studies highlight the application of the CRISPR/Cas9 system to target heat-sensitive genes for developing plant resistance against heat stress.

5. Improvement in Cold Stress Tolerance Using CRISPR/Cas System

Cold stress due to chilling and freezing temperatures hinders plant growth and development. Low temperature inhibited plant metabolic activities, producing osmotic and oxidative stress [5][7]. Mechanical damage and metabolic dysfunction caused by freezing temperatures reduced plant growth and development. In A. thaliana, the two subclasses, namely DREB1/CBF and DREB2, are induced by cold and dehydration, respectively [4][21]. Expression of T. aestivum TaICE41 and TaICE87 in transgenic A. thaliana activated the expression of COR genes and consequently led to the enhancement of cold tolerance, but only after cold acclimation [181][87]. The overexpression of AtDREB1A under the RD29A promoter conferred increased drought and freezing tolerance to transgenic A. thaliana plants without affecting growth and development [182][88]. Several studies have demonstrated that WRKY transcription factors are essential in cold, heat, drought, and salinity stress [183][89]. In Cucumber (Cucumis sativus), CsWRKY46 is a WRKY transcription factor that confers cold resistance in transgenic plants by controlling cold-stress responsive genes in an ABA-dependent manner. Overexpression of CsWRKY46 regulates freezing and chilling resistance and increases the expression of stress-inducible genes, including RD29A and COR47 [184][90]. In strawberries (Fragaria vesca), FvICE1 is a positive regulator of cold and drought resistances, and the overexpressed FvICE1 gene improved cold and drought tolerance at the phenotypic and physiological levels. Mutant (fvice1) generated using the CRISPR/Cas9 system demonstrated lower tolerance to cold and drought.
The C-repeat binding factors (CBF) are highly conserved CBF cold-response-system components in many plant species. It has a major role in cold acclimation and freezing tolerance in response to low temperatures. CRISPR–Cas9-mediated SlCBF1 mutagenesis reduced chilling tolerance of L. esculentum because of higher electrolyte leakage, increased indole acetic acid contents, decreased abscisic acid, methyl jasmonate, and down-regulated CBF-related genes [186][91]. Similarly, CRISPR–Cas9-mediated mutagenesis of CGFS-type GRXs (SlGRXS14, SlGRXS15, SlGRXS16, and SlGRXS17) genes showed the sensitivity of Slgrxs mutants to various abiotic stresses as compared to wild-type in L. esculentum [187][92]. Plant annexins are Ca2+-dependent phospholipid-binding proteins that play a role in development and protection from environmental stresses. CRISPR/Cas9-mediated knockout mutant of annexin gene OsAnn3 decreased cold tolerance in O sativa [188][93]. OsMYB30 confers cold sensitivity by interacting with an OsJAZ9 protein and downregulating the expression of β-amylase genes in O. sativa [189][94]. Novel mutants were generated by simultaneously editing three genes, OsPIN5b (panicle length gene), GS3 (grain size gene), and OsMYB30, using the CRISPR–Cas9 system showed higher yield and excellent cold tolerance [190][95]. PYR1]/PYR1-like [PYL]/regulatory components of the ABA receptor detects abscisic acid during abiotic stress. CRISPR/Cas9 technology was used to edit PYL1–PYL6 and PYL12 (group I) and PYL7–PYL11 and PYL13 (group II) genes of O. sativa [191][96]. A knockout mutant of the OsPRP1 gene of O. sativa generated by CRISPR/Cas9 demonstrated less antioxidant enzyme activity and accumulated lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to wild-type plants under low-temperature stress [192][97]. CRISPR/Cas9-mediated base editing technology generated the point mutations in two genes (OsWSL5 and OsZEBRA3) in protoplasts and regenerated plants of O. sativa. OsWSL5 encodes a novel chloroplast-targeted pentatricopeptide repeat protein essential in rice chloroplast biogenesis under cold stress [193,194][98][99]. These studies indicated the potential of CRISPR/Cas9–mediated mutagenesis in developing resistance to chilling and freezing temperatures in drought-tolerant cultivars by knocking out or overexpressing target genes through precise genome editing.

6. Improvement in Metal and Herbicide Stress Tolerance Using CRISPR/Cas System

Heavy metals, including arsenic (As), copper (Cu), cobalt (Co), cadmium (Cd), iron (Fe), manganese (Mn), nickel (Ni), zinc (Zn), mercury (Hg), lead (Pb) have accumulated in soils as a result of various human activities such as the overuse of agricultural chemicals (fertilizer, herbicides, and pesticides), improper disposal of industrial and sewage waste [11][100]. Heavy metals are responsible for causing oxidative stress in plants and generate reactive oxygen species, leading to cellular injury. CRISPR-Cas9-mediated mutagenesis was used to improve heat metal stress in plants. OXP1 is one of the enzymes involved in 5-oxoproline metabolism and the pathway for glutathione recycling. The oxp1/CRISPR tolerated plants tolerated heavy metals, such as Cd and amisulbrom (a sulfonamide) [13][101]. Cadmium stress activates the antioxidant defense system and increases the production of abscisic acid (ABA), glutathione (GSH), salicylic acid (SA), jasmonic acid (JA), and nitric oxide (NO) [195,196][102][103]. Absorption of Cd by the roots is mediated by O. sativa genes (OsNramp1, OsCd1, and OsNramp5). In O. sativa, OsHMA2, OsCCX2, and CAL1 regulate Cd transport to the xylem, and OsHMA3 negatively regulates Cd xylem loading. Manipulation in the expression of these genes through CRISPR/Cas9 can minimize the Cd concentration in O. sativa [197][104]. CRISPR/Cas9 knockout mutants of OsLCT1 and OsNramp5 exhibited reduced levels of Cd in O. sativa [198][105]. OsNRAMP1 modulates metal ion and reactive oxygen species (ROS) homeostasis. Osnramp1 mutants generated through CRISPR/Cas9 displayed reduced levels of heavy metals (Cd and Pb) in leaves and grains of O. sativa [14][106]. The OsHAK1 gene encodes a Cs+-permeable K+ transporter that regulates the absorption and translocation of cesium [Cs+] in O. sativa. CRISPR/Cas9 knockout mutant of OsHAK1 exhibited a significant reduction in Cs+ uptake in O. sativa [199][107]. Potassium [K+] is a critical macronutrient for plant growth and development. ROS was strongly induced and accumulated in K+-deficient plants. Gene Prxs have been involved in the toxic reduction and intracellular H2O2 scavenging. The overexpressed OsPRX2 gene improved tolerance to K+ deficiency by affecting stomatal movement in O. sativa [200][108]. OsARM1 (Arsenite-Responsive MYB1) is the R2R3 MYB transcription factor that regulates arsenic-associated transporters genes in O. sativa, and the knockout mutant (osarm1) generated using CRISPR/Cas system displayed improved tolerance to arsenic [201][109].
Herbicides destroy weeds and crop plants by interfering with or altering their metabolic processes, resulting in low yields. Thus, tolerance to herbicides can be one of the essential traits for crop plants that could improve farming practices and crop productivity. CRISPR/Cas-based genome editing techniques efficiently modify target genes and hold great potential in engineering plants with herbicide resistance [202][110]. In recent years, CRISPR-Cas9-based technology has been used to generate herbicide-tolerant crops, including O. sativa, Z. mays, and G. max [203,204,205,206][111][112][113][114]. Acetolactate synthase (ALS) catalyzes the step in the biosynthesis of the branched-chain amino acids, including leucine (Leu), isoleucine (Ile), and valine (Val). Enzyme ALS is the target enzyme for two classes of herbicides: sulfonylurea and imidazolinone. Tolerance to ALS-inhibiting herbicides has been developed using a genome editing system in A. thaliana, O. sativa, T. aestivum, Z. mays, S. lycopersicon, and Saccharum spp. [42,203,204,205,206,207,208][111][112][113][114][115][116][117]. Herbicide-resistant plants were generated through CRISPR/Cas9-mediated homologous recombination of ALS in O. sativa [204][112]. Similarly, chlorsulfuron resistance was enhanced in Z. mays by editing the ALS2 gene (substitution P165 with Ser) using either single-strand oligonucleotides or double-strand DNA vectors as repair templates [203][111]. Moreover, P171F substitution in the OsALS1 allele was performed in the O. sativa cultivar Nangeng 46 by precise base editing, resulting in tolerance to the herbicide bispyribac-sodium [208][117]. Four different missense mutations (P171S, P171A, P171Y, and P171F) in the P171 codon of the ALS gene showed different degrees of tolerance towards five typical herbicides (Sulfonylurea, imidazolinone, triazolopyrimidine, pyr-imidinyl-thiobenzoates, and sulfonyl-aminocarbonyl-triazolinone) belongs to five chemical families of ALS inhibitors in O. sativa [206][114]. A novel allele (G628W) was developed from a G-to-T transversion at position 1882 of the OsALS gene and conferred resistance to herbicide stress. These mutants of rice plants conferred resistance to herbicides of imazethapyr (IMT) and imazapic (IMP) [209][118]. The CRISPR/Cas9 system was also successfully used to edit the ALS1 gene of G. max to obtain chlorsulfuron-resistant plants [207][116]. Mutation of the Proline-186 residue in the ALS gene conferred chlorsulfuron resistance in L. esculentum [210][119]. An enzyme of activation-induced cytidine deaminase (AID) converts C to U in DNA/RNA by deamination. A synthetic complex of nuclease-deficient Cas9 fused to an AID, which is target-AID enables targeted nucleotide substitutions (C to T or G to A). The point mutation C287T of the ALS gene in rice plants resisted the herbicide imazamox [211][120]. In T. aestivum, herbicide-tolerant plants were produced by nucleotide editing of the acetolactate synthase (ALS) gene and acetyl-coenzyme A carboxylase gene, which conferred resistance to sulfonylurea, imidazolinone-, and aryloxy phenoxy propionate-type herbicides [205][113]. Co-editing three copies of the ALS gene resulted in herbicide tolerance in Saccharum spp. [42][115].
Glyphosate is one of the well-known and broad-spectrum herbicides used in the weed management of resistant crops, such as C. annum, G. max, O. sativa, and Z. mays. Glyphosate inhibits the enzyme EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), involved in the biosynthesis of aromatic amino acids and secondary metabolites. Site-specific gene replacements and insertions in the rice endogenous EPSPS gene resulted in glyphosate-resistant plants [212,213][121][122]. The CRISPR/Cas9 tool creates a structural variation (genomic duplication or inversion) in chromosomes. The resulting mutant developed through CRISPR/Cas technology showed the increased accumulation of transcripts of CP12 and Ubiquitin2 genes and the 10-fold upregulated expression of HPPD (4-hydroxyphenyl pyruvate dioxygenase) and PPO1 (protoporphyrinogen oxidase) resulted in herbicide resistance without affecting the yield and other agronomically important traits in O. sativa [214][123]. CRISPR-Cas9 system was used to edit the target genes of herbicides (ALS and EPSPS) in L. esculentum cv. Micro-Tom [215][124]. Another herbicide resistance gene, Bentazon Sensitive Lethal (BEL), gives resistance to herbicides of bentazon and sulfonylurea in O. sativa. CRISPR/Cas9-based mutation of the BEL gene was evaluated in rice using the Agrobacterium-mediated stable transformation [216][125]. The efficiency of mutagenesis ranged from 2% to 16%, and the phenotypic analysis indicated that the mutated plant was sensitive to the herbicide bentazon. Precise editing of the endogenous α-tubulin homolog gene OsTubA2 using CRIPSR-mediated adenine base editors at the T1981 site. The point mutation in the OsTubA2 gene transformed the O. sativa cultivar into a herbicide (dinitroaniline) tolerant cultivar [217][126]. These studies summarised the application of CRISPR/Cas-mediated editing of genes responsible for metal and herbicide stress tolerance in plants.

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