Enhancing the productivity and resilience of rice (Oryza sativa) under environmental stress conditions using clustered regularly interspaced short palindromic repeats (CRISPR) technology
Aamir Riaz
A # * , Muhammad Uzair A # , Ali Raza B , Safeena Inam A , Rashid Iqbal C D , Saima Jameel A , Bushra Bibi A and Muhammad Ramzan Khan A *
A
B
C
D
# These authors contributed equally to this work
Handling Editor: Muhammad Nadeem
Abstract
Rice (Oryza sativa) is a crucial staple crop worldwide, providing nutrition to more than half of the global population. Nonetheless, the sustainability of grain production is increasingly jeopardized by both biotic and abiotic stressors exacerbated by climate change, which increases the crop’s rvulnerability to pests and diseases. Genome-editing by clustered regularly interspaced short palindromic repeats and CRISPR-associated Protein 9 (CRISPR-Cas9) presents a potential solution for enhancing rice productivity and resilience under climatic stress. This technology can alter a plant’s genetic components without the introduction of foreign DNA or genes. It has become one of the most extensively used approaches for discovering new gene functions and creating novel varieties that exhibit a higher tolerance to both abiotic and biotic stresses, herbicide resistance, and improved yield production. This study examines numerous CRISPR-Cas9-based genome-editing techniques for gene knockout, gene knock-in, multiplexing for simultaneous disruption of multiple genes, base-editing, and prime-editing. This review elucidates the application of genome-editing technologies to enhance rice production by directly targeting yield-related genes or indirectly modulating numerous abiotic and biotic stress-responsive genes. We highlight the need to integrate genetic advancements with conventional and advanced agricultural methods to create rice varieties that are resilient to stresses, thereby safeguarding food security and promoting agricultural sustainability amid climatic concerns.
Keywords: biotic, drought, genome editing, heat, heavy metals, mutation, rice, stresses, yield.
Introduction
Cereals constitute an essential part of the diets of the world’s growing population. Rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum) account for roughly half of the global calorie consumption for humans (Falcon et al. 2022). Rice is the most significant food crop for Asian countries, particularly in south-east Asia where it is cultivated on millions of hectares of land (Giller et al. 2021; Husain and Sharma 2022). It is the world’s second-most planted crop (Rahaman and Shehab 2019; Singh et al. 2021). Rice was cultivated 10,000 years ago in the river basins of south and south-east Asia and China (Zeng 2020), and is today, it is also grown Latin America, Europe, certain parts of Africa, and the United States (Samal et al. 2021).
There are various obstacles that need to be overcome in order to continue increasing rice production to meet the food needs of the world’s expanding population (Boyd et al. 2020; Islam et al. 2024). The migration of workers from rural areas to urban areas is limiting the labor supply available to work on farms, which places rice production at risk due to the crop’s high eliance on water and labor. The widespread use of high-yielding rice varieties and hybrids, combined with excessive fertilizer application, particularly nitrogen, has been the primary driving force behind the notable increase in rice production over the past 40 years (Yu et al. 2020; Tseng et al. 2021). Overuse of nitrogen fertilizer has resulted in environmental issues such as eutrophication of surface and groundwater, ozone layer depletion, and greenhouse warming. These issues have sparked widespread concern around the world (Chandini et al. 2019; Ashitha et al. 2021).
Overcoming diseases caused by bacteria, fungi, and viruses is another obstacle that is critical to maximizing yield potential (Pérez-Méndez et al. 2022; Raza et al. 2022; Chaitanya et al. 2023). The dynamics of rice diseases have changed through time as a consequence of changes in farming techniques, a reduction in varietal range that has led to a restricted genetic basis, and changes in climate (Heredia et al. 2022; Khoury et al. 2022). Certain diseases have become more prevalent and threatening, having spread to more locations. A great number of diseases that were previously regarded as relatively unimportant have recently emerged as major economic threats in certain areas. Because new and more dangerous viral strains have emerged, managing diseases has become much more difficult due to a faster breakdown of disease resistance. Therefore, new technologies are needed to keep pace with these changes. This review discusses the role of clustered regularly interspaced short palindromic repeats and CRISPR-associated Protein 9 (CRISPR/Cas9) system, a genome-editing tool, to develop rice varieties that are more resistant to range of abiotic and biotic stresses caused a changing climate (Fig. 1).
Responses to abiotic stresses in rice
Abiotic stressors that adversely agricultural production are often inter-related with one another, and often manifest in plants as osmotic stress, a failure in ion transportation, or a disruption in the metabolism of plant cells (Madani et al. 2019). These responses are induced by a collection of genes that alter how they are expressed, which has an impact on the rate of growth and ultimately, profitability. Therefore, the identification of genes that are sensitive to abiotic challenges is required to comprehend the processes that agricultural plants use to react when they are subjected to abiotic stresses (Bueno and Lopes 2020). Plants are are constantly adapting to improve their defense systems against various abiotic pressures .Here, we discuss some of these abiotic pressures.
Cold
Cold stress reduces the productivity of crops (Gull et al. 2019; Kul et al. 2019; Teklić et al. 2021) by affecting crop quality and fruit production, especially in temperate climates where chilling and freezing can detrimentally affect plant development and growth (Selvaraj et al. 2022). Plants can undergo acclimatization where they build up resistance to potentially damaging cold shocks to adapt to cold or freezing conditions (Sakina et al. 2019). Despite this, a significant number of key crops are not yet capable of acclimatization to cold conditions. The abiotic stress that is generated by low temperatures affects every element of the cellular functioning of plants. There are many different signal transduction pathways, such as elements of reactive oxygen species (ROS), protein phosphate, protein kinase, abscisic acid (ABA), and Ca2, which transduce the effects of cold stressors. Among these, ABA has been shown to be the most effective (Sakina et al. 2019).
Salt
Soil salinity is a worldwide threat to agriculture across the globe by lowering the agricultural production (Singh 2022). Salt stress has adverse effects on plant growth and production in two main ways: (1) osmotic load; and (2) oxidative stress, which is the imbalance in the production and neutralisation of reactive oxygen species (ROS) (Hussain et al. 2019; Adil et al. 2023). Due to the presence of excess salts, the osmotic pressure in the soil solution is higher than that in the plant cells during salt stress. As a result, the plant’s ability to absorb essential minerals and water, such as calcium and potassium is hampered, which induce downstream effect such as diminished cell function and growth, impaired cytosolic efficiency, and impaired assimilate synthesis (Singhal et al. 2021; El-Badri et al. 2022).
Drought
Average global temperatures and atmospheric carbon dioxide levels are both rising at a steady rate, which has resulted in widespread alterations to the planet’s climate (Isson et al. 2020). The shift in weather has resulted in an irregular rainfall distribution that contributes to drought in areas that once had regular precipitation (Wang et al. 2022a). Because of extreme drought conditions, the quantity of soil water that is available to plants has been reducing, which has led to the untimely mortality of plants (Frei et al. 2022). Plants stop growing in drought conditions as metabolic energy required for growth decreases (Frei et al. 2022). Under drought stress, plants produce protective chemicals by mobilizing compounds that are necessary for osmoregulation (Jacques et al. 2021).
Heat
A rise in temperature around the world has become a major cause for concern and affects plant development and yield (Hinojosa et al. 2019; Morales et al. 2020). When plants are subjected to heat stress, seed germination and photosynethetic effectiveness decrease. When a plant experiences heat shock during the productive growth stage, heat damages the tapetal cell’s functioning, and another cell becomes dysplastic (Younis et al. 2020).
Heavy metals
Increased use of synthetic fertilizers, irrigation with sewage wastewater, and rapid urbanization have resulted in toxic metals being introduced into agricultural soils, which has had a negative impact on the soil–plant environment (Gull et al. 2019; Lin et al. 2022). Heavy metals like chromium (Cr), cadmium (Cd), lead (Pb), mercury (Hg), arsenic (As), and thallium (Tl) are harmful living organisms, and the contamination of rice grains with Cd and As is a worldwide problem (Yu et al. 2016; Sharma et al. 2022). Toxic levels of zinc (Zn) and Cd uptake can impact on iron (Fe) absorption in moderate soil dryness and cause detrimental effects on the photosynthesis process and yield (Table 1) (Slamet-Loedin et al. 2015; Özyiğit et al. 2021). The accumulation of heavy metals in rice can result in biomagnification within the food chain, posing a potential hazard to human and animal health.
| Heavy metal | Reported yield loss (%) | References | |
|---|---|---|---|
| Hg | 51–70 | Hillary and Ceasar (2022) | |
| Cr | 30–47 | AbdElgawad et al. (2023) | |
| Al | 25–60 | AbdElgawad et al. (2023) | |
| Ni | 53–69 | Aziz et al. (2015) | |
| Zn | 20–40 | Panhwar et al. (2015) | |
| Cu | 10–90 | Xu et al. (2006) | |
| Fe | 16–78 | Audebert and Fofana (2009) | |
| Cd | 34–63 | Kanu et al. (2017) | |
| As | 30–40 | Muehe et al. (2019) | |
| Pb | 46–69 | Ashraf et al. (2017) |
Biotic stresses and crops
Plants are exposed to a diverse range of biotic stressors, such as fungal infections, viral diseases, bacterial infections, insects, and nematodes that cause infections, and crop damage, ultimately affecting crop yield (Panth et al. 2020). Weed imposes significant impact on plant growth through the competition of resources, sunlight, and space. There are several pathogenic plants that have the ability to enter the host plant and infect both the roots and the aboveground parts of the host. Cuscuta sp., Arceuthobium sp., Orobanche sp., and Phelipanche aegyptiaca are parasitic plants that infest host plants, significantly damaging the agro-economic production (Ghosh and Dey 2022). Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo) (Oliva et al. 2019), is one of the most significant rice bacterial pathogens. Magnaporthe oryzae, the pathogen responsible for rice blast disease, has resulted in significant economic losses in rice production (Wei et al. 2024). Viruses are obligate parasites with a strong capacity to infect plants, resulting in a significant decrease in crop output (Das et al. 2019). The CRISPR-Cas technique provides plants with resistance against both DNA and RNA viruses by directly targeting and cleaving the viral genome (Mushtaq et al. 2020). The fungal infection can readily infiltrate the host plant due to its genetic adaptability. Fungi are responsible for causing various diseases, such as mildew, smut, and rot, which ultimately lead to significant reductions in crop yield (Dong and Ronald 2019).
Genome-editing technologies
Due to a new era of genome engineering and genome-editing technologies, plant genomes may now be altered rapidly, precisely, and efficiently. In the 1990s, mega nucleases (MNs) and zinc-finger nucleases (ZFNs) established the foundation for genome-editing and sparked the advancement of this science (Bibikova et al. 2002; Carroll 2021). MNs were a newly discovered type of nucleases that were commonly employed in the editing of plant genomes. MNs are differentiated from other endonucleases by their substantial recognition site, often consisting of 12–40 base pairs (bps). This characteristic makes MNs the most effective method for delivering all types of vectors, including plant RNA viruses (Mishra and Zhao 2018). The most frequent MNs are homing endonucleases. MNs pose challenges for modification when used alongside other genome-targeting methods due to the frequent fusion of their DNA-binding and catalytic domains, which cannot be easily separated (Zhang et al. 2010). Prior research in several crops shown the potential utility of modified MNs for genetic editing in plants. Nevertheless, due to the challenging nature of managing MNs, further investigation is required to enhance this approach. Consequently, scientists have focused their efforts on developing gene engineering techniques that are more efficient, uncomplicated, and precise. These include ZFNs, transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats and CRISPR-associated Protein 9 (CRISPR/Cas9).
ZFNs revolutionized genome modification by being the pioneering protein reagents with the ability to precisely target specific locations. ZFNs act as DNA-binding domains that precisely identify and bind to a specific target spot by perfectly recognizing three base pairs (Rai et al. 2019). They are a highly effective and influential tools for genome-editing due to their ability to target double-strand breaks (DSBs) specifically (Durai et al. 2005). ZFNs consist of two essential domains: (1) the DNA-binding domain; and (2) the DNA cleavage domain. These domains are necessary for ZFNs to carry out its function (Papworth et al. 2006). According to (Carlson et al. 2012), the DNA-binding domain consists of 300–600 zinc-finger repeats, each that can recognise and interpret nine to 18 bps of DNA sequence. The DNA cleavage domain is an extensively acknowledged element of the type II restriction endonuclease Fok1, serving as a non-selective cleavage domain. According to (Carroll et al. 2006), this identical domain also functions as the DNA cleavage domain in ZFNs. ZFNs have been extensively created and tested across multiple species. In rice, low starch levels, dwarf morphologies, and no SSIVa mRNA expression were found in transgenic plants expressing premature stop codons and substitution events, which were created by ZFN-mediated targeted gene disruption in the SSIVa rice gene’s coding sequence (Jung et al. 2018). The occurrence of unintended consequences decreased as technology grew more precise and efficient.
Similar to ZFNs, TALENs are site-directed mutagenesis enzymes found in the rice plant pathogenic bacteria X. oryzae. In 2010, TALENs were created by fusing a transcription activator-like effector (TALE) with FokI’s catalytic domain. Genome-editing became accessible to scientists because they were easier to utilize (Christian et al. 2010). The DNA binding domain of TALENs consists of a single TALE protein that binds to a single base pair of DNA. Researchers were able to decode the recognition preferences of 400 distinct RVDs in 2014, which established TALENs as a significant tool for studying gene function and gene therapy (Yang et al. 2014). TALEs are distinguished by their 34-amino-acid repetitive sequences, which enable precise editing of a single base pair (Zhang et al. 2019a). Non-adjacent repetitive sequences can impact TALE proteins as well. TALE proteins continuously repair the nucleotides of the DNA sequence at the thymidine base located at the 5′ end. Without a 50 T, the functions of TALE transcription factors (TALE-TFs) and TALE recombinase (TALE-R) are diminished (Lamb et al. 2013). TALENs surpass ZFNs in terms of mouldability and exhibit a reduced off-target rate. Nevertheless, TALENs and ZFNs have challenges in protein synthesis, validation, and design, which serve as obstacles to their widespread implementation for standardized purposes.
CRISPR/Cas is an adaptive immune system found in bacteria and archaea that defends against viruses and plasmids (Barrangou et al. 2007). The CRISPR/Cas system comprises two primary components: (1) the CRISPR array; and (2) the Cas proteins (Westra et al. 2014; van Beljouw et al. 2023). The CRISPR array is a segment of the bacterial genome that consists of brief recurring sequences interspersed with distinct spacer sequences obtained from the genetic material of prior intruders, such as viruses or plasmids. Its purpose is to retain a record of past infections. The Cas proteins, also known as CRISPR-associated proteins, play a crucial role in identifying and cutting foreign genetic material. They operate by utilising the data stored in the CRISPR array to detect and selectively target particular regions in the foreign DNA or RNA (Atia et al. 2024). The CRISPR/Cas immunity pathway comprises multiple sequential stages. The CRISPR/Cas9 system was created in 2012 as a tool for editing genomes and has since become the most commonly employed technology for this purpose (Mali et al. 2013). The CRISPR/Cas9 system consists of two primary components: (1) Cas9, an RNA-guided DNA endonuclease; and (2) a single guide RNA (sgRNA). The CRISPR-Cas9 system identifies specific DNA sequences by means of DNA–RNA interaction. The Cas9 protein requires guidance to the target location provided by the sgRNA, which consists of 20 nucleotides that are complementary to the target sequences (Jiang et al. 2013a; Shan et al. 2013).
CRISP/Cas differs from ZFN and TALEN genome-editing methods as it identifies target DNA sequences through DNA-protein interaction. The CRISPR/Cas9 method necessitates the creation of a sgRNA, which may be adjusted for various genomic targets through the simple alteration of the complementary sequence (Jiang et al. 2013a). Cas9 cleaves the DNA strand at the spot where the sgRNA binds to the protospacer-adjacent motif (PAM) sequence, which is a short nucleotide sequence located near the cutting site at the 3′ end of the target region.
As previously mentioned, the CRISPR/Cas9-mediated targeted DSB can be repaired using either error-prone non-homologous end joining (NHEJ) or precise-mutation homology-directed repair (HDR). For HDR, accurate genetic changes can be made by using DNA repair donors that have homology arms. Nevertheless, achieving high-fidelity HDR while minimising the occurrence of NHEJ remains a significant obstacle (Miyaoka et al. 2016). The CRISPR-Cas9 system has undergone additional optimisation since its initial development, resulting in improved specificity and the ability to do precise genome-editing while minimising any impact on the genome (Jaganathan et al. 2018). Therefore, enhancements to CRISPR/Cas9 that reduce off-target effects and/or eliminate the protospacer adjacent motif (PAM) constraints can broaden its range of uses. Specifically, several methods have been demonstrated to effectively reduce unintended consequences, such as modifying Cas9 and sgRNA, conducting bioinformatics analysis, and optimising delivery methods (Gohil et al. 2021). However, various strategies for adapting have been developed through research to counteract the negative effects of biotic stressors. One method to mitigate the consequences of these pressures on crops is to investigate the genetics of the agents responsible for biotic and abiotic stressors in plants. Using genome-editing to develop resistant varieties of agricultural plants has proven to be an effective strategy for reducing the damaging effects of both biotic and abiotic pressures on plant (Knorre and Vlasov 1985; ul Haq et al. 2019).
According to Lu et al. (2018), the use of CRISPR/Cas9 technology for genome-editing in rice crop is a promising alternative to conventional breeding methods (Fig. 2) (Lu et al. 2018). Genome-editing is a scientific technique for modifying or regulating specific DNA sequences within a genome. CRISPR/Cas9 is a molecular tool that can be precisely engaged to make desirable modifications in targeted locations within complex genomes. According to Lu et al. (2018), the use of CRISPR/Cas9 technology for genome-editing in rice offers a promising alternative to conventional breeding methods (Fig. 2) (Hsu et al. 2014). In response to insect attacks on rice grains, researchers were able to produce CRISPR-based genetically modified (GM) rice in 2018 by suppressing the synthesis of serotonin, a neurotransmitter found in mammals (Lu et al. 2018) to develop rice gains that are resistant to planthoppers and stem borers (Chen et al. 2011; Iftikhar et al. 2023).
CRISPR systems for rice crop improvements
Enhancing plant resistance to biotic and abiotic stresses while increasing yield is the main goal of agricultural research (Delorge et al. 2014; Rehman et al. 2022; Rauf et al. 2024). Recently, more accurate methods for editing the genome have been created, replacing older techniques like random mutagenesis, naturally occurring mutations, and plant classical breeding. These conventional methods are time-consuming and inefficient in producing individuals with the desired characteristics. Emerging gene methodologies have become crucial instruments in the field of plant science and plant molecular breeding (Nazir et al. 2022). The progression of genome-editing technology has gone through multiple phases, including the use of ZFNs, TALENs, and the more recent development of the CRISPR/Cas system (Nazir et al. 2022; Dong 2024). CRISPR/Cas system is known for its versatility, simplicity, and cost-effectiveness, and has been the dominant method for genetic manipulation in the field of genome-editing since its discovery. Genome-editing technology has been extensively utilized for gene functional study and crop enhancement. The initial reports of rice and other plant genome-editing using CRISPR/Cas technology date back to 2013 (Jiang et al. 2013b; Miao et al. 2013; Xie and Yang 2013). Shan et al. (2013) created two sgRNAs to target the rice phytoene desaturase gene OsPDS and achieved a 15% mutagenesis efficiency in transformed rice (Nipponbare) protoplasts. In a study conducted by Jiang et al. (2013a), single guide RNAs (sgRNAs) were employed to manipulate the genes OsSWEET14 and OsSWEET11. These genes play a crucial role in the immune response to bacterial blight induced by X. oryzae. Following protoplast transformation, the presence of the desired modifications was verified using PCR and sequencing. Following the validation of CRISPR/Cas genome-editing in rice with the publishing of these results, numerous studies have been conducted to implement it for various applications (Fig. 3).
Visual presentation shows the CRISPR systems used for improvement in rice plants. The road map and timeline show the CRISPR systems along with targeted genes improved by these tools: CRISPR/SpCas9 (Shan et al. 2013); CRISPR/SpCas9-VQR (Hu et al. 2016); CRISPR/SpCas9-VRER (Hu et al. 2016); CRISPR/SaCas9 (Kaya et al. 2016)); CRISPR/FnCpf-1 (Endo et al. 2016); CRISPR/LbCpf-1 (Wang et al. 2017); CRISPR/AsCpf-1 (Tang et al. 2017a); CRISPR/LbCpf-1-RR (Li et al. 2018); CRISPR/LbCpf-1-RV and CRISPR/FnCpf-1-RR; and CRISPR/FnCpf-1-RVR (Zhong et al. 2018).
Applications of CRISPR/Cas in rice
This method has several interesting applications, one of which is the development of a genome-wide mutant library developmet (Fig. 4). This library may be utilized for determining the roles of genes and for improving genetics. Meng et al. (2017) combed through the rice genome in search of every possible sgRNA. They did this while keeping in mind that the seed region (12 bps immediately next to PAM) must only correlate once somewhere in the genome. In the exon regions, they discovered 1,535,852 target locations in 52,916 genes of rice. sgRNAs that are placed in exons that are close to the start of ORFs are chosen, and two sgRNAs from each candidate gene are picked. This is done so that loss-of-function mutations may be enriched. This process results in the production of 12,802 genes and 25,604 sgRNAs. Through the process of sequencing, the plasmid library’s quality, sgRNA accuracy, and gene coverage are evaluated. The Agrobacterium is then used for rice (cv. Zhonghai 11), resulting in the creation of over 14,000 distinct T0 lines. 182 T0 plants are arbitrarily sequenced to assess the accuracy and comprehensiveness of the mutations, with 139 of them having a single precise sgRNA. A total of 139 plants were found to have a single accurate sgRNA, with 136 of them being unique, implying that each sgRNA was well represented. Furthermore, potential off-target effects for specific sgRNAs were investigated, but no off-target modifications were discovered (Meng et al. 2017). Lu et al. (2017) employed a similar technique to the one described earlier to create a sgRNA library with 88,541 members that targeted 34,234 genes. The rice cultivar MSU7 has an average of 2.59 sgRNAs per gene (Lu et al. 2017). A sequencing process is used to validate the library’s creation. As a result, over 90% of the plasmid was confirmed and 99% of the genome was examined. Following that, the plasmid is transferred into Agrobacterium for rice (MSU7, ‘Rice Genome Annotation Project’ MSU version 7) modification. The mutation rate is 83.9%, resulting in the creation of 84,384 transgenic plants. A high-throughput genotyping procedure is used (Bell et al. 2014). This method was used on 9216 plants, of which 7004 were successfully identified and 86.4% had a sgRNA. This resulted in the identification of 5541 plants and the analysis of 2326 loci (Lu et al. 2017).
Different breeding and genome-editing methods and their timeframes required to produce improved rice varieties.
CRISPR/Cas technology is an influential tool for developing mutant libraries in rice, which can significantly speed up the breeding progression. Furthermore, it can help with the functional description of unidentified genes in rice and other crop plants. A notable and exciting application of the CRISPR/Cas system is the accurate removal of marker genes in genetically modified plants (Khan et al. 2021).
Biosafety is a critical factor in determining whether or not selection marker genes code for herbicide resistance and/or antibiotic resistance. Selection markers can also make the insertion of additional genes into transgenic lines more problematic. Consequently, eliminating and inserting selection markers should be prioritized. Two short guide RNAs were generated to target opposing ends of the GUS gene in the B1 transgenic rice (cv. Nipponbare) line in order to achieve this objective (Nandy and Srivastava 2012; Srivastava 2021). Using this technique, only five of 113 transgenic events showed evidence of the expected complete gene deletion. Excisions occur on both alleles in two of these events. When the sequence data for these events was examined, it was discovered that the excision was quite precise, as no mutations were found in either of the two cleavage sites (Srivastava et al. 2017). This newly discovered use of CRISPR/Cas has the potential to be utilized for the excision of selectable marker genes in rice and other plant species, thus enhancing breeding strategies (Fig. 4).
Strategies for high-yielding environmental stress resistant rice crop through CRISPR technology
To improve germplasm traits such as yield, the conventional breeding process includes selecting parental lines, crossbreeding, and multiple selection cycles, which can take up to seven to eight growing seasons to release a new variety. In contrast, CRISPR/Cas9 technology can accomplish this in less than 1 year (Hussain et al. 2018), and has been used to modify several negative regulator genes, resulting in enhanced yield and other significant agronomic characteristics. Being the foremost grain crop, rice has emerged as a prominent central focus for the CRISPR-based genome engineering technology, with the aim of enhancing its inherent characteristics. This method enables the mutation of several genes and quantitative trait loci (QTLs) in rice plants, with the aim of enhancing grain yield and other essential traits (Zihe et al. 2019; Riaz et al. 2021). Tables 2–5 show the genes of rice plants, that have been edited by CRISPR/Cas9.
| Improved gene | Traits improved | Chromosomal number | MSU ID | Reference | |
|---|---|---|---|---|---|
| SD2 | Tiller number | – | – | Cui et al. (2020) | |
| SCM1 | Stem cross-sectional area, Tiller number | 1 | – | Cui et al. (2020) | |
| SCM2 | Stem cross-sectional area, Tiller number | 6 | LOC_Os06g45460.1 | Cui et al. (2020) | |
| OsGA20ox2 | Reduced gibberellins, Reduced flag leaf length, Grain yield | 3 | LOC_Os03g63970.1 | Han et al. (2019) | |
| SD1 | Resistance to lodging, grain yield | 1 | LOC_Os01g66100.1 | Hu et al. (2019) | |
| OsPYL9 | Grain yield under limited water accessibility | 6 | LOC_Os06g33690.1 | Usman et al. (2020) | |
| PYL1 | Grain yield, grain number | 10 | LOC_Os10g42280.1 | Miao et al. (2018) | |
| PYL4 | Grain yield, grain number | 1 | LOC_Os01g61210.1 | Miao et al. (2018) | |
| PYL6 | Grain yield, grain number | 3 | LOC_Os03g18600.1 | Miao et al. (2018) | |
| OsBADH2 | Grain size, grain yield, aroma | 8 | LOC_Os08g32870.1 | Nawaz et al. (2020) | |
| OsFWL | Number of tillers, grain size, number of cells in flag leaf grain length, flag leaf area, grain yield | 2 | LOC_Os02g52550.1 | Gao et al. (2020) | |
| DEP1 | Grain size, grain number, grain yield, panicle architecture | 9 | LOC_Os09g26999.1 | Ma et al. (2015) | |
| GS3 | Grain size, grain number, grain yield, panicle architecture | 12 | LOC_Os12g34380.1 | Ma et al. (2015) | |
| Gn1a | Grain size, grain number, grain yield, panicle architecture | 1 | LOC_Os01g10110.1 | Ma et al. (2015) | |
| OsSPL16 | Grain size, grain yield | 8 | LOC_Os08g41940.1 | Usman et al. (2021) | |
| GW2 | Grain weight, grain yield | 2 | LOC_Os02g14720.1 | Xu et al. (2016) | |
| GW5 | Grain weight, grain yield | 5 | LOC_Os05g09520.1 | Xu et al. (2016) | |
| GW6 | Grain weight, grain yield | 6 | LOC_Os06g15620.1 | Xu et al. (2016) | |
| OsPIN5b | Grain size, length of pinnacle | 8 | LOC_Os08g41720.1 | Zeng et al. (2020) | |
| IPA1 | Number of tillers, grain yield, plant architecture, grain size, dense erect panicles, grain number | 8 | LOC_Os08g39890.1 | Schausberger (2018) | |
| SBE1 | Starch metabolism | 6 | LOC_Os06g51084.1 | Romero and Gatica-Arias (2019) |
| Disease/pathogen | Improved gene | Pathogen resistance/disease | Chromosome No. | MSU ID | References | |
|---|---|---|---|---|---|---|
| Fungi | OsERF922 | Magnaporthe oryzae/Rice blast | 1 | LOC_Os01g54890.1 | Schausberger (2018) | |
| OsALB1 | Magnaporthe oryzae/Rice blast | 10 | LOC_Os10g38950.1 | Foster et al. (2018) | ||
| OsUSTA | Ustilaginiodea virens/False smut | 10 | LOC_Os10g38950.1 | Liang et al. (2018) | ||
| OsUvSLT2 | Ustilaginiodea virens/False smut | 10 | LOC_Os10g38950.1 | Liang et al. (2018) | ||
| OsPi21 | Magnaporthe oryzae/Rice blast | 4 | LOC_Os04g32850.3 LOC_Os04g32850.1 LOC_Os04g32850.2 | Nawaz et al. (2020) | ||
| OsRSY1 | Magnaporthe oryzae/Rice blast | 10 | LOC_Os10g38950.1 | Foster et al. (2018) | ||
| Bacteria | OsSWEET11 | Xanthomonas oryzae pv. oryzae/Bacterial leaf blight | 8 | LOC_Os08g42350.1 | Kim et al. (2019) | |
| OsSWEET13 | Xanthomonas oryzae pv. oryzae/Bacterial leaf blight | 12 | LOC_Os12g29220.1 | Oliva et al. (2019) | ||
| OsSWEET14 | Xanthomonas oryzae pv. oryzae/Bacterial leaf blight | 11 | LOC_Os11g31190.1 | Xu et al. (2019) | ||
| Os8N3 | Xanthomonas oryzae pv. oryzae/Bacterial leaf blight | 8 | LOC_Os08g42350.1 | Kim et al. (2019) | ||
| OsXa13 | Xanthomonas oryzae pv. oryzae/Bacterial leaf blight | 8 | LOC_Os08g42350.1 | MacNeil et al. (2020) | ||
| Virus | eIF4G | Rice tongro spherical virus | 7 | LOC_Os07g36940.1 | Macovei et al. (2018) | |
| General disease resistance | OsMPK1 | Infection resistant signaling pathway | 6 | LOC_Os06g06090.1 | Minkenberg et al. (2017) | |
| OsMPK2 | Infection resistant signaling pathway | – | – | Minkenberg et al. (2017) | ||
| OsMPK5 | Infection resistant signaling pathway | 3 | LOC_Os03g17700.1 | Xie and Yang (2013) | ||
| OsMPK6 | Infection resistant signaling pathway | 10 | LOC_Os10g38950.1 | Minkenberg et al. (2017) | ||
| Herbicide resistance | OsEPSPS | Glyphosate | – | – | Li et al. (2016) | |
| OsALS | Bispyrabic-sodium, Imazamox, Imazethapyr-IMT and Imazapic-IMP, Nicosulphuron, Pyroxasulam, Flucarbazonesodium | 2 | LOC_Os02g30630.1 | Malhotra et al. (2016), Shimatani et al. (2017), Kuang et al. (2020) and Zhang and Gao (2020) | ||
| OsFTIP1 | Imazamox | 6 | LOC_Os06g41090.1 | Shimatani et al. (2017) |
| Stress | Improved gene | Chromosome number | MSU ID | Reference | |
|---|---|---|---|---|---|
| Drought | OsSAPK2 | 7 | LOC_Os07g42940.1 | Lou et al. (2017) | |
| OsSRL1 | 7 | LOC_Os07g01240.1 | Liao et al. (2019) | ||
| OsSRL2 | 3 | LOC_Os03g19520.1 | Liao et al. (2019) | ||
| OsPYL9 | 6 | LOC_Os06g33690.1 | Usman et al. (2020) | ||
| OsERA1 | 1 | LOC_Os01g53600.1 | Ogata et al. (2020) | ||
| DST | 3 | LOC_Os03g57240.1 | Santosh Kumar et al. (2020) | ||
| OsmiR535 | – | – | Yue et al. (2020) | ||
| Salinity | OsSAPK2 | 7 | LOC_Os07g42940.1 | Lou et al. (2017) | |
| OsRR22 | 6 | LOC_Os06g08440.1 | Zhang et al. (2019a) | ||
| DST | 3 | LOC_Os03g57240.1 | Santosh Kumar et al. (2020) | ||
| OsmiR535 | – | – | Yue et al. (2020) | ||
| Cold stress | OsAnn3 | 5 | LOC_Os05g31750.1 | Shen et al. (2017) | |
| OsMYB30 | 2 | LOC_Os02g41510.1 | Zeng et al. (2020) |
MSU ID and chromosome numbers are derived from (https://shigen.nig.ac.jp/rice/oryzabase/quicksearch/list).
| Heavy metal | Targeted gene | Chromosome number | MSU ID | Reference | |
|---|---|---|---|---|---|
| Cs | OsHAK1 | 4 | LOC_Os04g32920.1 | Nieves-Cordones et al. (2017) | |
| Mn, Cd, Fe, Pb | OsNramp5 | 7 | LOC_Os07g15370.1 | Tang et al. (2017b) | |
| Cd | OsLCT1 | 6 | LOC_Os06g38120.1 | Songmei et al. (2019) | |
| Cd | OsLCT2 | 6 | – | Tang et al. (2022) | |
| Cd, As | OsNRAMP1 | 7 | LOC_Os07g15460.1 | Chang et al. (2020) | |
| Cd | OsABCG36 | 1 | LOC_Os01g42380.1 | Fu et al. (2019) | |
| Si, As | OsLsi | 6 | LOC_Os06g12310.1 | Hillary and Ceasar (2022) |
Biotic stress tolerance in rice by CRISPR
It is estimated that biotic stressors, which include infections caused by bacteria, viruses, and fungi are responsible for 20–40% of the total losses in agricultural production across the globe (Gimenez et al. 2018; Moustafa-Farag et al. 2020; Bhoi et al. 2022). Repetitive stress exposures have caused significant yield loss in plants over time, which has had a severe impact on the food secuity worldwide (Richard et al. 2022). Scientists have used CRISPR technology to develop rice crops that are resistant to a variety of pathogenic microorganisms (Chen et al. 2019; Karmakar et al. 2022) (Table 3).
Fungal infections, particularly powdery mildew, frequently impede crop productivity and can be disastrous. Another fungal disease that can have a significant impact on agricultural productivity is rice blast. The OsSEC3A and OsERF922 genes were targeted by researchers using the CRISPR/Cas9 technology in order to significantly boost resistance to rice blast infection (Wang et al. 2016a; Ma et al. 2018). The OsERF922 mutant did not exhibit any alterations in its agronomic traits. However, researchers observed an increase in SA content in OsSEC3A, which led to a reduction in its size (Ma et al. 2018). The partial resistance to rice blast can also be induced by CRISPR/Cas9-mediated mutations of rice Bsr-d1 and Pi21 genes. However, the magnitude of this effect is comparatively weaker than that of OsERF922 (Nawaz et al. 2020; Zhou et al. 2022).
According to Schloss and Handelsman (2004), numerous bacterial species have the potential to induce disease in plants, with several of them manifesting various disease symptoms (Schloss and Handelsman 2004). The management of plant pathogenic bacteria is complicated due to difficulties in early disease detection and insufficient pesticides. However, the application of the CRISPR/Cas9 approach in plant genome modification has been shown to enhance crop resistance to bacterial diseases. Susceptibility is linked to the OsSWEET13 gene, which encodes a sucrose transporter and plays a significant character in plant–pathogen interaction. X. oryzae produces an effector protein called PthXo2 in affected plants, which triggers the expression of OsSWEET13 in the host, leading to susceptibility in rice. Resistance against bacterial blight was increased in rice plants by suppressing the OsSWEET13 promoter (Zhou et al. 2015).
Abiotic stress tolerance in rice by CRISPR
Salinity, drought, heavy metals, and extreme temperature exposure are all examples of abiotic stresses that have an important impact on plant development and growth (Table 4). These factors have the potential to reduce crop production by up to 50% (Liu et al. 2022; Nawaz et al. 2023; Noman and Azhar 2023). According to research, salinization has affected 7% of the world’s land and 20% of its arable land, and there are signs that the problem will worsen (Al Murad et al. 2020). The activation of osmotic stress, ion stress, and secondary stress pathways comes about as a consequence of plants being subjected to salt stress (Yang and Guo 2018). This occurrence has been observed to have a negative influence on crop yield and quality (Siddiqui et al. 2017; Ali et al. 2024). It has been found that using CRISPR/Cas9 technology to cause mutation in the OsRR22 gene increases salt tolerance in rice (Zhang et al. 2019b; Han et al. 2022), while other agronomic traits remained unchanged. Using CRISPR/Cas9 technology, Liu et al. (2020) were able to effectively alter the OsRAV2 gene (Liu et al. 2020). Researchers successfully mutated the OsRAV2 gene using CRISPR/Cas9 technology, resulting in a mutant rice crop with increased survival rates when subjected to salt stress. Furthermore, using CRISPR/Cas9 technology to disrupt genes like OsDST, OsNAC041, and OsmiR535 has been shown to significantly improve rice’s ability to tolerate salt-induced stress. Ogata et al. (2020) discovered that using CRISPR/Cas9-mediated mutagenesis on OsERA1 led to an important enhancement in drought stress tolerance in rice (Wang et al. 2016b; Ogata et al. 2020; Santosh Kumar et al. 2020; Yue et al. 2020). CRISPR-based genome-editing technology has been used to improve the rice plant’s ability to withstand extreme temperatures, both cold and heat. Specifically, using this technology, the OsAnn3 gene in rice was mutated to confer tolerance to cold stress in the plant (Kumar et al. 2022).
Resistance to heavy metal contamination in rice by CRISPR
The negative consequences of heavy metals on plant growth and yield pose a significant barrier to agricultural sustainability. Stresses such as oxidative stress, osmotic ion imbalance, membrane tissue instability, cell death, and disrupted metabolic equilibrium detrimentally impact on plant physiology and biochemistry (Hoque et al. 2021). The ingestion of plants that have accumulated heavy metals can result in significant harm to human and animal health (Kaur et al. 2021). A previous study found that using CRISPR/Cas9 technology to knock down the OsLCT1 and OsNramp5 genes reduced Cd accumulation in rice (Table 5). Another study found that disrupting OsNRAMP1 with CRISPR/Cas9 reduced the levels Cd and Pb in rice grains (Chang et al. 2020; Chu et al. 2022). Simultaneously, it was discovered that OsNRAMP5 and OsNRAMP1 have different abilities to reduce Cd accumulation. The CRISPR/Cas9 method was used to prevent arsenic absorption and transportation in rice by disrupting the R2R3 MYB transcription factor OsARM1. Furthermore, Nieves-Cordones et al. (2017) used the CRISPR/Cas9 technique to inactivate the K+ transporter OsHAK1 to produce rice plants with lower cesium (Cs) levels (Nieves-Cordones et al. 2017; Chang et al. 2020). Researchers employed CRISPR/Cas9 technology to create oslcd single mutants derived from both indica and japonica rice crops. In addition, we created single mutants of osnramp5 and double mutants of oslcd, and osnramp5 in the indica background. When cultivated in paddy soils contaminated with Cd, all single mutants of oslcd acquired a lower amount of Cd compared to the wild-types (Chen et al. 2023). Li et al. (2022) findings suggest that CRISPR/Cas9 mutated OsPMEI12 plays a role in controlling the process of methyl esterification during growth, impacting the composition of the cell wall and several agronomic features. Additionally, it is likely to have a significant function in responding to phytohormones and Cd stress. The application of CRISPR/Cas9 resulted in a higher accumulation of Cd in rice straws, seeds, and brown rice due to mutation. Conversely, the overexpression lines exhibited comparatively lower quantities of Cd. The findings indicate that the metal chaperone gene OsHIPP56 can suppress the harmful effects of cadmium by decreasing its accumulation in crops. In summary, our work contributes to the comprehension of the functional significance of OsHIPP56 in rice’s detoxification of Cd (Zhao et al. 2022).
Limitations
CRISPR/Cas9 has multiple applications in plant breeding, although it does have specific constraints. The primary obstacle is in the limited availability of a narrow gene pool regulating vital phenotype. Therefore, the accessibility of genes is vital for this tool. CRISPR/Cas9 still has relatively few restrictions (Fig. 5), even though it has numerous benefits and a wide range of applications. However, there are a few imminent challenges that might impede the effectiveness of the CRISPR/Cas9 system in the creation of disease resistance. These challenges are in Fig. 5.
SpCas9 necessitates a 50-NGG-30PAM in close proximity to the 20 nt DNA target sequence because to its exclusive recognition of NGG PAM sites. This constraint restricts its efficacy. While this restriction is crucial, the objective is to deactivate the gene using selected mutagenesis in any circumstance. However, PAM restricts the editing of resistance genes. Hence, it is necessary to select sequences that are located near PAM (Mohamed et al. 2024). It is possible that the direct manipulation of S genes would result in a loss of fitness owing to the S genes’ connection with other desired genes, in particular, the genes that govern plant development and growth. In addition, the mutation in the targeted gene might cause the route of the product to become disrupted, which would eventually have a negative impact on a large number of other products that use the same pathway. This cost to fitness is not as devastating as the previous one, but it still has the potential to result in a lack of key micronutrients and phenotypic abnormalities. Nevertheless, the cost of viability may vary due to the targeting of specific gene/s, and it is also dependent on various other parameters, like disease susceptibility facilitators, defense silencers, genes, or post relevant aspects in the reporter gene (van Schie and Takken 2014). Research on naturally occurring or synthetically produced pathogen inactivating promoters or transcription factors has been the primary focus of scientists during the past two decades (Rushton et al. 2002). By employing regulatory elements, it is possible to develop pathogen-inducible CRISPR systems. These systems may transiently knock off a S gene that corresponds to a particular micronutrient or a sugar promoter, so avoiding the fitness repercussions that would otherwise occur. Editing may be used to create desirable S gene mutants in almost all plants that are of interest for breeding, bypassing species boundaries in the process. It is anticipated that new S genes will be identified, which will result in the availability of additional sites for genome-editing.
‘Off-target mutations’ is major factor that cause hindrance (Hahn and Nekrasov 2019). Off-target genome-editing refers to genetic changes occurring at unplanned and non-specific regions. This type of editing can occur due to erroneous gRNA or in a gRNA-independent manner (Jin et al. 2019). CRISPR/Cas can cause random off-target variations in the plant DNA. The lack of ability to effectively extract Cas proteins and generate transgene-free crops has impeded the commercialization of CRISPR-edited crops (Wang et al. 2022b). The majority of the work that has been done to solve the issue of off-target editing can be broken down into two categories: (1) development of a mechanism for off-target detection; and (2) engineering of the CRISPR mechanism for high-quality modification. To date, a variety of bioinformatics tools have been created to address this problem. Some examples of these tools are CasOFFinder and CCTop (Koo et al. 2015), as well as DISCOVER (Wienert et al. 2019). Because each analytical instrument has its own set of benefits and drawbacks, researchers may need to choose an analytical method that is appropriate for the task. In contrast, great progress has been made in reducing the off-target activity of CRISPR/Cas9, which is an encouraging development. Cas9 proteins with enhanced target selectivity have to be created first. These Cas9 proteins include eSpCas9 (Slaymaker et al. 2016), Sniper Cas9 (Lee et al. 2018), HF-Cas9 (Kleinstiver et al. 2016), and HypaCas9 (Chen et al. 2017). Sniper Cas9 was selected from a collection of SpCas9 mutants that displayed improved specificity, while HypaCas9, HiFiCas9, and eSpCas9 were produced by rational design for structural alterations to boost specificity. The majority of the modified Cas proteins exhibited a striking drop in off-target levels while maintaining their activity on the intended target. In addition, improvements in specificity have been made through the creation of gRNA (Moon et al. 2019). Recent studies have demonstrated the occurrence of off-target mutations in rice as a result of cytosine base-editing. Jin et al. (2019), but not adenine-based editors. This finding demonstrates that the new tools also need some kind of modification.
Both the safety of people and other living species, as well as questions about commercialization, are closely tied to these concerns. Because the subsequent transgenic does not contain any foreign nuclei, the crops evolved through transgene-free mechanisms would not be labeled as transgenic, and it would be easier to adopt them for commercial cultivation. This is because the TKC and VIGE systems do not involve the use of any foreign material. However, the widespread use of genome-edited crops presents a difficult ethical conundrum. Concerning this issue, certain states have openly adopted genome-edited crops and a significant number of nations are presently engaged in debates surrounding this topic. A European court has ruled that the crop with changed genome is considered genetically modified. Despite the fact that the decision has certain technical limitations, which may be reviewed by member states, there is still a significant opportunity for the creation of new breeds and varieties quickly and effectively (Abbott 2018). It is anticipated that the use of this technology would unquestionably go a long way toward creating conditions that would facilitate the acceptance of genetically modified crops in the majority of nations in a manner that is comparatively straightforward.
In the past, the CRISPR/Cas9 system has been used to alter RNA and DNA based viruses and it has assisted researchers in the production of plant material that is resistant to viruses. However, the efficacy of this mechanism has been called into doubt since it has been shown to be ineffective against the viral immunity found in eukaryotes. Based on the results of Mehta et al. (2019), we may hypothesize that CRISPR/Cas9 has significant limits not just against DNA viruses but also RNA viruses, and these restrictions are caused by the viruses’ ability to escape and rapidly replicate. As a result, there is an immediate need for the creation of a version of CRISPR that is efficient, effective, and socially acceptable, such as CRISPR/Cas13a. This will allow for the resolution of problems caused by viruses. CRISPR/Cas13a has tremendous ability as just a precise, robust, and expandable RNA-targeting channel for RNA manipulation techniques and RNA virus targeting, according to recent findings (Aman et al. 2018). These results advocate that among the three Cas13 protein families (Cas13a, Cas13b, and Cas13c), CRISPR/Cas13a holds the most promise as an RNA-targeting platform. Cas13a enzymes have the potential to be repurposed as tools for a variety of applications, including the detection of nucleic acids, the suppression of RNA in mammalian and plant cells, and the monitoring of transcripts (Abudayyeh et al. 2017; Rehman et al. 2022). RNA interference with Cas13a may occur even without the biological protospacer-f-linking sequence (PFS). This finding is interesting as in the not-too-distant future, Cas13 could prove to be a superior option to CRISPR/Cas9 when it comes to targeting viral RNA. CRISPR-based-editing holds the capacity to enhance crops by increasing their ability to withstand abiotic and biotic stress and improving their nutritional composition. The CRISPR-based genome-editing technique is widely recognized as a highly potent technology for enhancing agriculture to sustain the constantly expanding population.
Future perspective
CRISPR technology has revolutionized agriculture by addressing specific crop problems and providing significant solutions to important agricultural issues. CRISPR/Cas9 technology is being employed to improve the quality, yield, and resilience of both monocots and dicots by precisely editing crop genomes. This approach enables the modification of specific genes, enhancing the plant’s ability to withstand biotic and abiotic stresses, ultimately leading to more robust and productive crop varieties. Rice is one of the world’s most important food crops, playing an imperative role in global food security. In light of increasing challenges posed by climate change and population growth, significant efforts are focused on developing new rice varieties that exhibit enhanced agronomic traits, particularly resilience to various biotic and abiotic stresses. The CRISPR/Cas9 system has emerged as a groundbreaking tool in this endeavor, offering unparalleled efficiency, simplicity, and versatility to precisely improve various crop traits. CRISPR/Cas technology has rapidly advanced, leading to DNA-free genome-editing systems, Cas9 variants, multi-gene targeting, and enhanced Homology-Directed Repair. Recent computational techniques have amplified its effectiveness, driving its growing adoption in real world applications.
CRISPR/Cas9 technology enables targeted rice genome modifications, enhancing adaptability and productivity, contributing to global food security in a rapidly changing climatic era. The regulatory frameworks around genetically modified organisms (GMOs) continue to be a major issue, irrespective of the considerable potential of CRISPR/Cas9 in rice enhancement. Although some regulatory concerns may be addressed by the capacity to modify genes without introducing foreign DNA, discussions over the implications of gene-editing in agriculture will continue to determine the technology’s potential uses. Communicating CRISPR/Cas-mediated genetic modification to the public is crucial for agriculture’s future. Establishing legal distinctions between gene-edited plants with foreign genes and those without is essential. Exemptions from regulations can facilitate easier integration. Clear regulations are crucial for the marketing approval of transgene-free, genome-edited crops, especially in regions where such practices are not yet prevalent. Advanced genome-editing techniques, like CRISPR/Cas9, hold significant promise for agricultural innovation; however, the lack of well-defined regulatory frameworks is widening the gap between laboratory advancements and practical field applications. To facilitate the safe and effective deployment of these technologies, comprehensive risk assessments, regulatory oversight, and precautionary measures are essential. Furthermore, optimizing transformation procedures is vital for the successful integration of CRISPR/Cas genome-editing into crop improvement initiatives. Community and private sector facilities can bridge the gap between scientific breakthroughs and practical agriculture by supplying advanced genome-editing tools, streamlining crop development processes, and fostering public acceptance of genome-edited crops through the implementation of science-based regulations. Our review will be useful in understanding the latest technical advancements, knowledge gaps, and challenges with technical adaptations that are necessary for the optimal usage of genome-editing techniques for crop development.
Compliance with ethical standards
This manuscript does not contain any studies with humans/animals performed by any authors.
Declaration of funding
This work is supported by the Ministry of National Food Security and Research of Pakistan released for the PSDP-project ‘Sino-Pak Agricultural Breeding Innovations Project for Rapid Yield Enhancement’ (857).
Author contributions
AAR and MRK designed the study. MU, AR, SI, BB, SJ, RI, and AAR data collection, data analysis, and writing of the original draft. MU and MRK revision of the article. MRK supervised the research. All authors review and edit the manuscript.
Acknowledgements
The authors acknowledge the research facilities provided by the Functional Genomics and Bioinformatics Group, National Institute for Genomics and Advanced Biotechnology (NIGAB), Pakistan.
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