CRISPR-Cas technology secures sustainability through its applications: a review in green biotechnology

Abstract

The CRISPR-Cas system's applications in biotechnology offer a promising avenue for addressing pressing global challenges, such as climate change, environmental pollution, the energy crisis, and the food crisis, thereby advancing sustainability. The ever-growing demand for food due to the projected population of around 9.6 billion by 2050 requires innovation in agriculture. CRISPR-Cas technology emerges as a powerful solution, enhancing crop varieties, optimizing yields, and improving resilience to stressors. It offers multiple gene editing, base editing, and prime editing, surpassing conventional methods. CRISPR-Cas introduces disease and herbicide resistance, high-yielding, drought-tolerant, and water-efficient crops to address rising water utilization and to improve the efficiency of agricultural practices which promise food sustainability and revolutionize agriculture for the benefit of future generations. The application of CRISPR-Cas technology extends beyond agriculture to address environmental challenges. With the adverse impacts of climate change and pollution endangering ecosystems, there is a growing need for sustainable solutions. The technology's potential in carbon capture and reduction through bio-sequestration is a pivotal strategy for combating climate change. Genomic advancements allow for the development of genetically modified organisms, optimizing biofuel and biomaterial production, and contributing to a renewable and sustainable energy future. This study reviews the multifaceted applications of CRISPR-Cas technology in the agricultural and environmental fields and emphasizes its potential to secure a sustainable future.

Introduction

Genes determine all of an organism's traits and characteristics and provide both an individual organism and a species with its unique identity. Nearly everything that has an impact on our lives, from our health to the food we consume is influenced by genes. So, to sum up a long narrative, genes create life. Many scientists believe that genome editing holds the secret to solving every issue that man has ever faced. A meticulous gene editing method seemed too ambitious and difficult a short while ago, but today, thanks to significant advancements in molecular biology, gene editing not only does not seem ambitious and sophisticated but also quite simple and approachable.

Nowadays, a sound molecular tool that is widely known as a defensive tactic in archaea and bacteria, broadly uses for genome editing and cell engineering. that is to say, CRISPR-Cas9 system which stands for “clustered regularly interspaced short palindromic repeat—CRISPR associated protein9”. The very first clue of the existence of this repetitious DNA sequence was announced by a group of researchers at Osaka University over 1987 during a genome analyzation of Escherichia coli. Later, in the next researches similar patterns of the genome were found in other bacteria and archaea (Ishino et al. 2018; Shivram et al. 2021). Back then, the true functionality of these DNA sequences was still unrevealed due to the lack of sufficient knowledge about DNA sequences, mainly mobile elements. For the revelation of the CRISPR-Cas system’s function the key question that had to be answered was the identification of the linkage between the Cas proteins and CRISPRs. Many researchers initially suggested that these two are engaged in DNA repair mechanism. Great headways in molecular biology, structural biology, bioinformatics, and generally biology disclosed that the CRISPR system is a crucial component of prokaryotic adaptive immunity which protects them from the attack of foreign DNA and invaders like bacteriophages, viral DNA, and plasmids. Over a viral infection, bacteria attain a small piece of the foreign DNA using enzymes such as Cas1-Cas2 and integrate it in between the palindromic repeated sequences to form CRISPR arrays. The acquired sequence of foreign DNA or spacer originated from Mobile Genetic Elements (MGEs) like bacteriophages, plasmid or transposons. By developing CRISPR arrays, bacteria will retain a memory to speak of former infections and trigger adaptive immunity in the cell (van der Oost et al. 2009; Ishino et al. 2018).

Studies show that this system exists in 40% and 90% of bacterial and archaeal genome respectively (Li et al. 2016). CRISPR-Cas9 systems are ubiquitous among prokaryotes based on a genomic analyze in the CRISPRdb online database which demonstrates that 1302 out of 2762 archaeal and bacterial strains have CRISPR arrays (Grissa et al. 2007). Cas9 protein which is one of the major parts of this system, as an RNA-reliant DNA-endonuclease, has the potential to cleaving any targeted sequence in the double-stranded DNA (dsDNA) with the help of two bacterial molecules named CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Cas9 portion of the Streptococcus pyogenes CRISPR-Cas9 system, include 2 domains named histidine-asparagine-histidine (HNH) and RuvC, which are in charge of dsDNA cleavage (Li et al. 2016).

In bacteria, the crRNA and tracrRNA are distinct molecule entities. The first step toward exploiting CRISPR-Cas9 system as a programmable tool for genome editing in eukaryotic cells had taken when scientists realized that a linker can link crRNA and tracrRNA to form a specific molecule called single guide RNA (sgRNA). sgRNA, which offers target specificity, also can be described as a chimeric and synthetized fusion of a CRISPR RNA with a bacterial origin (crRNA) and a trans-activating crRNA (tracrRNA) (Shanmugam et al. 2020). If the sgRNA complexes with a cas9 protein, it can cleave DNA just as a three-component system in bacteria (Cas9, crRNA, tracrRNA). Cas9 protein will cut any intended dsDNAs matched with the designed sgRNAs and together, it cuts 3–4 nucleotides upstream of the PAM. Cas9 recognizes the PAM sequence 5′-NGG, cleaves at situated close to the PAM and introduces a blunt-ended double-stranded break (DSB) (Cong and Zhang 2015; Puchta 2017; Li and Xia 2020). PAM sequence helps the Cas9 protein to interact with the DNA. Then, the DSBs may be repaired by the homology-directed repair (HDR) pathway and/or non-homologous end joining (NHEJ) pathway. These two repair pathways of DNA break both induced by CRISPR/Cas9. HDR pathway mostly used for precise allele or gene replacement or in other word “knock-in”, meanwhile, the NHEJ pathway is used for knock-out mutagenesis by making accidental indels (Mao et al. 2019; Li and Xia 2020). Commonly, a viral vector or a plasmid is used to transfect the projected cells with CRISPR-Cas9 machinery. This system could easily be engineered by some methods to precise gene edition in prokaryotes and eukaryotes including plant, animal, and microbial cells. It is easily possible to determine any sequence of about 20bp as a target for editing and all that has to be done is synthesizing the appropriate sgRNA complemented to the desired in inserting it into the cell along with the Cas9 (Cong and Zhang 2015).

The CRISPR system was initially identified as a genetic component of prokaryotes, but in recent years, scientists have shown a strong interest in the system, leading to the creation of multiple cost-effective applications, such as genome editing, that can be applied to a variety of eukaryotic cells. The edition of the genome can play a crucial part in solving numerous issues in a variety of fields, such as issues in the medical, agricultural, and environmental fields (Kozovska et al. 2021). Formerly, the most famous genome editing tools included transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs), but the emerging of CRISPR-Cas9 system as an easy-to-use, accurate, fast, and cost-efficient tool changed the game and started a revolution not only in genome edition but also for imaging, gene regulation and expression, DNA repair, chromatin engineering, and epigenetic editing (Doudna and Charpentier 2014; Adli 2018; Javed et al. 2018; Zaychikova et al. 2020). In addition, the CRISPR system enables genome manipulation at multiple sites (Wolter et al. 2019). Because of these properties, CRISPR-Cas9 systems can be exploited for genome engineering in any intended cells which shows the vital role of CRISPR-Cas systems in modern biotechnology (Manghwar et al. 2020).

For the purpose of genome editing, some new CRISPR systems have been developed and emerged in recent years like Cas12a/Cpf1 nuclease, Cas12b/C2c1 nuclease, Cas13/C2c2 nuclease, Cas14/Cas12f nucleases, and CasMINI to overcome the limits that CRISPR-Cas9 machinery may has (Biswas et al. 2021). All the different CRISPR-Cas systems have their own properties and differences for instance differences in size, PAM regions, and cleavage sites (Liu et al. 2020b). For instance, Cas12a improves the efficacy of NHEJ-based gene insertion and offers further benefits in knock-in strategies rather than Cas9. Cas12b is smaller than both Cas9 and Cas12a and offers more adaptability in versatile gRNA engineering (Liu et al. 2020b). In 2021, Stanford’s researchers by applying RNA and protein engineering techniques to the 529 amino acids Cas12f, generated a compact and efficient protein as functional as Cas12a but in reduced size and called it CasMINI. This system accelerates the various applications that CRISPR-Cas systems offer in different fields of biotechnology including gene editing and therapy (Xu et al. 2021).

Although there are many different perspectives on what biotechnology is, it is commonly recognized to be the utilization of organisms and biological systems to create new goods and efficient industrial, medical, and technical processes. Since advanced biotechnology mostly relies on the technology of recombinant DNA and genetic manipulation, the CRISPR-Cas technology has the potential of major impacts on biotechnological processes and is a chief tool for synthetic biology (Amin et al. 2011; Sampson and Weiss 2014; Zhang et al. 2020a). Biotechnology as a multidisciplinary major is divided into various subdivisions and each subdivision represents a specific area of biotechnology activities. Each area denotes a specific color, for instance, red for medical biotechnology, green for environmental and agricultural biotechnology, black or dark for malevolent affairs such as bioterrorism, anti-crop warfare, bio-warfare and bio-crime, blue for marine biotechnology, etc. (DaSilva 2004).

Green biotechnology refers to the application of biotechnological techniques for agricultural and environmental concerns to address those difficulties (DaSilva 2004; Bauer 2005). It is undeniably necessary to use effective and readily available biotechnology tools, such as CRISPR-Cas technology, to mitigate our environmental and agricultural problems to meet the growing demand for food, achieve sustainable agriculture, and combat the negative effects of climate change on our agriculture and environment (Fig. 1) (Karavolias et al. 2021; Tripathi et al. 2022a). The objective of this study is to review the CRISPR-Cas system's applications in green biotechnology and explain how this novel molecular technology might help us address pressing issues like climate change, environmental pollution, the energy crisis, the food crisis, etc., ultimately leading us in the direction of sustainability.

Fig. 1.

Applications of CRISPR-Cas technology positively impact agriculture and environment and mitigate the impacts of climate change

CRISPR-Cas leads us through sustainable agriculture

Sustainable agriculture is described as long-term, sustainable methods that provide the population with high-quality food and fiber to meet their needs and make efficient use of resources to improve people's quality of life. There are many challenges along the road to sustainable agriculture such as climate change, environmental pollution, shortage of water resources, salinization, land degradation, etc. (Harwood 2020). While accessible water and farmlands are being depleted, studies predict that the global population will reach 9.6 billion by 2050, increasing demand for crops and agricultural products by 60%. Population growth, climate change-induced disasters, and environmental pollution are just a few examples of negative events that might affect plant and crop output (Ricroch et al. 2017; Zhu et al. 2020; Wada et al. 2020). To revolutionize agriculture for the benefit of future generations, the technology that applies to genetic modification must speed up the domestication process and provide genetic diversity because of the aforementioned ever-increasing demand for food and the fact that only 15 of a total of 30,000 edible plants provide approximately 70% of the calories needed by humans. For this purpose, CRISPR-Cas technology offers quick crop varieties improvement (Khan et al. 2019). Research into CRISPR-Cas reveals that this technology possesses the potential to revolutionize existing agricultural methods, enhancing our capacity to ensure food security and effectively tackle various challenges (Zhu et al. 2020).

Increasing agricultural production and crop yield is beneficial and can be crucial in some countries, so using cutting-edge, affordable, innovative, and precise tools like CRISPR-Cas9 as a genome editing device that allows most crops and plants to be modified and improved is promising to overcome such concerns by optimizing and ameliorating crop yield, metabolic pathways, and biotic and abiotic resistance (Chen et al. 2019; Capdeville et al. 2023). CRISPR-Cas systems can optimize crops’ qualitative and/or quantitative traits so as to meet their growing worldwide demand (Scheben and Edwards 2018). All the mutations and genome editions introduced by CRISPR-Cas systems are hereditary. CRISPR-Cas9 which applied in many plants over the past years has the potential for simultaneous gene editions of multiple targets. Gene targeting (GT), is another approach for this system by which plants’ genomes can be engineered precisely (Wada et al. 2020). GT in plant and agricultural biotechnology mostly relies on HDR. However, CRISPR-Cas-based technologies like base-editing and prime editing are alternative methods to HDR as they do not require DSB and donor DNA and are more efficient methods (Zhu et al. 2020).

Today, a potent alternative method for conducting plant functional genomics studies is made possible by the ability to create targeted knockout lines using CRISPR technology. Due to the high efficiency of CRIPSR-mediated mutagenesis in plants, true-breeding mutant lines can be established for later characterization or employed in high-throughput functional screening (Jacobs et al. 2017). For instance, Pooled CRISPR libraries were transferred into a tomato to generate collections of mutant lines with minimum transformation attempts and in a reasonably short amount of time. The benefit of such a method would be the ability to produce a large number of mutants from a single transformation experiment, boosting the efficiency of creating knockout lines that could be applied to functional research (Jacobs et al. 2017).

The CRISPR-Cas9 system already has been successfully employed for the genome edition of various model plants like Arabidopsis, ornamental flowers, and crops such as apple, grape, potato, tomato, banana, peanut, maize, rice, soybean, sorghum, wheat, and tobacco, etc. for valuable plants production and plant studies. Although the use of this system has the potential of leading to sustainable agriculture, broad use of this system for agricultural plants and food production is still under debate like other gene editing technologies due to the unknown challenges and ethical risks it may pose to human, livestock and environment from the viewpoint of social science and some concerns like off-target effects (Bartkowski et al. 2018; Shu et al. 2020).

Crop improvement

Improvement is required to make crops more resistant to the deleterious and unprecedented impacts of climate change and related environmental stresses. Due to the dire need for enhanced food security, genes involved in crop improvement are the main target of gene editing in the field of agricultural biotechnology. For example, the CRISPR/Cas9 system edits the genome using single-sgRNAs, making it a simple, fast, and powerful tool for targeted gene mutagenesis, knock-in and knockout replacement, and transcriptional regulation (Hussain et al. 2018; Young et al. 2019; Zhang et al. 2021a). The use of a CRISPR-Cas9 system in crop editing has enabled advancements in plant breeding, providing precision and ease of editing, as well as lower costs (Chen et al. 2019; Gleim et al. 2020). Recently, bacterial type II CRISPR/Cas systems have been extensively used and have accomplished numerous goals in the field of agricultural research such as improving biotic and abiotic stress in crops, yield enhancement, herbicide resistance, disease resistance, and nutritional enrichment (Fig. 2). This system has significant properties that allow modification of a specific genome sequence, and researchers are interested in using this system for genome editing. The site specificity for complementary base pairing is provided by crRNA. Following base attachment to a tracrRNA, the target DNA sequence is cut by Cas9 endonuclease, which is guided by crRNA. The CRISPR/Cas9 technique has been used in model plants such as Arabidopsis and Nicotiana benthamiana for genome editing, as well as crops (such as wheat, rice, and sorghum) through temporary or constant transformation. The CRISPR/Cas9 system will be used to create epigenetic changes in plants, which may ameliorate crop yield and resistance to various diseases (Khan et al. 2018; Jaganathan et al. 2018; Rasheed et al. 2021).

Fig. 2.

Many experiments have been conducted on different crops genome using CRISPR-Cas9 technology for the aim of crop improvement. This promising technique introduces high-yield and resistance products against biotic and abiotic stresses that accelerates the pathway to meet the sustainable agriculture goals

Disease resistance

Many food crop species, including rice, tomato, wheat, barley, soybean, etc., have undergone several successful genetic modification trials using the CRISPR-Cas9 technology to create resistance plants against biotic stresses and diseases. Many plant diseases can significantly reduce the crop yield. For instance, Bacterial blight in rice is a biotic stress factor caused by Xanthomonas oryzae pv. Oryzae and can decrease the production of rice by 20%-50%. The fungus Magnaporthe oryzae is also another pathogen of rice which causes another biotic stress called rice blast and decreases rice production by 10–35% (Li et al. 2019; Zhang et al. 2021b).

Pseudomonas syringae pv. tomato DC 3000, which generates coronatine and interacts with the co-receptor JAZ2 to stimulate stomata opening and promote bacterial leaf colonization, causes bacterial speck disease in tomatoes (Solanum lycopersicum). A dominant allele of JAZ2 lacking the C-terminal Jas domain was created by editing SlJAZ2 using CRISPR-Cas9. This variant of JAZ2 limits stomatal reopening and offers resistance against bacterial speck disease (Ortigosa et al. 2019). An Arabidopsis gene called DMR6 (DOWNY MILDEW RESISTANT 6) has a strong association with salicylic acid regulation and, consequently, plant pathogen infection. Targeting SlDmr6-1, its tomato ortholog, with CRISPR has given the plant broad-spectrum resistance against numerous diseases, including Xanthomonas spp., P. syringae, and Phytophthora capsica (Tripathi et al. 2021).

One of the most significant bacterial infections in terms of economic impact is citrus canker caused by Xanthomonas citri subsp, for which CRISPR offers a resistance option. One of the most destructive citrus diseases, citri, reduces yields in citrus-growing regions all over the world. PthA4, the primary TALE (transcription activator-like effector nuclease) of the bacterium, attaches to the effector-binding site in the promoter of the LATERAL ORGAN BOUNDARIES 1 (CsLOB1) susceptibility gene and promotes the gene's expression to aid in the development of citrus canker. Two separate studies found that CRISPR-mediated editing of CsLOB1 considerably lessens the symptoms of citrus canker in two citrus species, grapefruit, and Wanjincheng orange (Wang et al. 2019a; Jeong et al. 2019).

Many more genes have been targeted related to disease resistance in plants via CRISPR-Cas technology which shows the application of this spectacular molecular system to improve crops resistance against pathogens (Table 1). This application of CRISPR-Cas brings us food security and leads us toward sustainable agriculture.

Table 1.

CRISPR-Cas and disease resistance. disease resistance was achieved through the genetic modification of several plant species using the CRISPR-Cas system

Crop Species	Disease name	Target	Type of manipulation	Type of CRISPR-Cas	Result	References
Musa acuminata	Banana Xanthomonas wilt (BXW)	MusaDMR6	Knockout	CRISPR-Cas9	enhanced resistance to BXW	Tripathi et al. (2022b)
Hordeum vulgare	Bipolaris leaf blight	HvMORC1, HvMORC6a	Knockout	CRISPR/Cas9	higher immunity attained against the disease	Galli et al. (2022)
Oryza sativa L	Bacterial blight	Os8N3	Knockout	CRISPR-Cas9	enhanced resistance to Xanthomonas oryzae pv. oryzae	Kim et al. (2019)
Solanum lycopersicum cv. Money Maker	Tomato yellow leaf curl (TYLC)	rgsCaM	Overexpression	CRISPR-Cas9	reducing TYLCV titer in tomato plants	Ghorbani Faal et al. 2(020)
Gossypium hirsutum	verticillium wilt	Gh14-3-3d	Knock-in	CRISPR/Cas9	Enhanced resistance to verticillium wilt	Zhang et al. (2018)
Vitis vinifera	Powdery mildew	VvMLO3	Knockout	CRISPR/Cas9	Enhanced resistance to Powdery mildew	Wan et al. (2020)
Ocimum basilicum L	Downy Mildew	ObHSK	Knockout	CRISPR/Cas9	Enhanced resistance to Downy Mildew	(Zhang et al. 2021c)
Oryza sativa L	Bacterial blight	Adding EBEAvrXa23 in the promoter region of xa23 allele	Knock-in	CRISPR/Cas9	enhanced resistance to Xanthomonas oryzae pv. oryzae	Wei et al. (2021)
Capsicum annuum L	Anthracnose	CaERF28	Multiple indels at the target site	Single Transcript Unit – CRISPR/Cas9	Enhanced resistance to anthracnose	Mishra et al. (2021)
Oryza sativa L	Rice blast	Pi21, OsERF922	Frame-shift gene mutations	CRISPR/Cas9	Resistant to rice blast	Chattopadhyay et al. (2022)
Gossypium hirsutum	Fusarium wilt	GhGLR4.8	Single nucleotide mutation	CRISPR/Cas9	Resistant to Fusarium wilt	Liu et al. (2021c)
Theobroma cacao	black pod disease	TcNPR3	Knock-out	CRISPR/Cas9	Resistant to black pod disease	Etaware (2021)
Oryza sativa L	rice dwarf disease	double strand RNA genome of Southern rice black‐streaked dwarf virus (SRBSDV)	Induction of RNases activity to cleave viral RNA	CRISPR/Cas13a	plant develops strong resistance to SRBSDV	Zhang et al. (2019)
Zea mays L	rough dwarf disease	Zm00001d010255	Knock-out	CRISPR/Cas9	Resistant to rough dwarf disease	Wang et al. (2022b)
Citrus sinensis	citrus canker	CsPDS	the ninth exon of CsPDS edited	CRISPR/LbCas12a	Resistant to citrus canker	Jia et al. (2019)
Solanum lycopersicum	bacterial canker	SlPR-1	transcriptional activation of the intended gene	CRISPRa	Enhanced resistance to bacterial canker of tomato	García-Murillo et al. (2023)
Citrullus lanatus	Fusarium wilt	Clpsk1	Knock-out	CRISPR/Cas9	Resistance against Fusarium oxysporum f.sp. niveum	Zhang et al. (2020b)
Vitis vinifera	grey mould	VvWRKY52	Knock-out	CRISPR/Cas9	Resistance of the plant to Botrytis cinerea	Wang et al. (2018)

Herbicide resistance

To improve agricultural efficiency, herbicide resistance is one of the most vital traits to induce in a plant (Gherekhloo et al. 2017). CRISPR-Cas systems suggest an innovative solution to introduce herbicide-resistant plants. Much research on many plant species has been conducted by CRISPR-Cas9 to induce herbicide-resistant traits like in maize, soybean, rice, etc. (Table 2) (Tian et al. 2018; Wu et al. 2020). The primary resistance mechanism is target site mutation, which has been successfully used to create herbicide-resistant germplasm in many crop species. Choosing target genes associated with important resistance traits is vital for creating plants that are herbicide-resistant. Notably, the ALS, ACCase, and EPSPS genes have made a significant number of candidate genes for creating herbicide-resistant crops (Dong et al. 2021). Through the NHEJ pathway, the CRISPR/Cas9 system can also produce effective intron-mediated site-specific gene substitution and insertion. Glyphosate resistance in goosegrass is caused by double amino acid substitution (T102I and P106S) in the conserved motif of the endogenous EPSPS gene (Gherekhloo et al. 2017). Also, new rice germplasms with resistance to bispyribac-sodium were generated by inserting point mutations at the 548th and 627th amino acid positions of the rice ALS gene (Butt et al. 2017). To improve HDR performance in plants, a chimeric Cas9-VirD2 protein was developed. VirD2 is a Viral protein that cleaves the bottom strands of the Ti plasmid on the left and right border (Wang et al. 2021b). Additionally, the precise OsALS allele alteration that leads to herbicide-resistant rice was accomplished using this approach (Zafar et al. 2023). Likewise, by introducing a donor template and a geminiviral replicon into plant cells, the glyphosate-resistance trait in rapeseed was improved (Wu et al. 2020).

Table 2.

CRISPR-Cas and herbicide resistance. Through the genetic alteration of various plant species utilizing the CRISPR-Cas system, herbicide resistance has been attained

Crop species/ plant models	Target	Transfer method	Type of manipulation	Type of CRISPR-Cas	Result	References
Setaria italica	SiFMBP, SiBADH2, SiALS, SiIPK1, SiACC, SiDof4, SiGBSS1	Agrobacterium-mediated transformation	Single and multi-gene knockout	CRISPR/Cas9 + base editors (CBE and ABE)	Mutant plant resistant to herbicides introduced	Liang et al. (2022)
Arabidopsis	OXP1	-	indel mutations	CRISPR/Cas9	Tolerance to heavy metals observed, and sensitivity to sulfamethoxazole decreased	Baeg et al. (2021)
Oryza sativa L	OsPUT1, OsPUT2, OsPUT3	Agrobacterium-mediated transformation	Knockout	CRISPR/Cas9	Sensitivity to paraquat decreased	Lyu et al. (2022)
Zea mays L	ALS1, ALS2	-	base editing	CRISPR/Cas9	resistance to sulfonylurea herbicides increased	Wang et al. (2022b)
Citrus sinensis	CsALS	Agrobacterium-mediated transformation	base editing	CBE fused to nSpCas9	Resistance to imazapyr increased	Alquézar et al. (2022)
Citrullus lanatus	ClALS	Agrobacterium-mediated transformation	base editing	CRISPR/Cas9-mediated base-editing	Resistance to sulfonylurea herbicides increased	Tian et al. (2018)
Oryza sativa	OsALS	Agrobacterium-mediated transformation	base editing	CRISPR-Cas9	Resistance to Bispyribac Sodium increased	Zafar et al. (2023)
Marchantia polymorpha	MpPAM16	Agrobacterium-mediated transformation	Loss of function	CRISPR-Cas9	Resistance to thaxtomin A increased	Casey et al. (2023)
Arabidopsis thaliana	ALS, AT3G48560	Agrobacterium-mediated transformation	introducing point mutation by GT	CRISPR/Cas12a	Resistanceto sulfonylurea and imidazoline increased	Wolter and Puchta (2019)
Citrus paradise	CpALS	Agrobacterium-mediated transformation	base editing	CRISPR/ nCas9 + CBE	Resistance to chlorsulfuron increased	Huang et al. (2022b)

Crop yield, quality, shape, and nutritional value enhancement

CRISPR-Cas technology has been used extensively in studies on a wide range of plant species to improve crop output, quality, shape, and nutritional value (Table 3). Yield is a multi-factor and complex trait and results in various phenotypic traits such as crops size, weight, and tiller number. After 2013, CRISPR-Cas9 system has extensively been utilized to optimize the traits related to the size and shape of the crop. For instance, in rice, GS3, Gn1a, GW2, GW5, TGW6, OsGS3, OsGW2, OsIPA, and OsGn1a are responsible for Grain length and width and also have targeted by CRISPR-Cas9 system in different experiments to increase the grain yield and were effective or in tomato several gene responsible for fruit size have been targeted via CRISPR-Cas9 (Chen et al. 2019; Chattopadhyay et al. 2022; Li et al. 2022a).

Table 3.

CRISPR-Cas mediated genetic modification to improve crop output, quality, shape, and nutritional value

	Crop species/ plant models	Target	Transfer method	Type of manipulation	Type of CRISPR-Cas	Result	References
Yield	Brassica napus	BnaMAX1	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	semi-dwarf and improved branching phenotypes observed. Compared to the wild type, yield per plant increased	Zheng et al. (2020)
	Oryza sativa	Os03g0603100, Os03g0568400, GL3.2, OsBADH2	Agrobacterium-mediated transformation	GT	CRISPR/Cas9	Grain yield and quality ameliorated	Usman et al. (2020a)
	Oryza sativa	OsPYL9	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Grain yield enhanced under both well-watered and drought conditions	Usman et al. (2020b)
	Triticum aestivum	TaGW2, TaGLW7 TaGW8, TaCKX2-1	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Grain size and weight increased	Hamdan et al. (2022)
	Triticum aestivum	TaGASR7	Vector inserted into protoplasts	Knock-out	CRISPR/Cas9	Grain wight increased	Zhang et al. (2016)
	Triticum aestivum	TaGW7-B1	Vector inserted into protoplasts	Deletion	CRISPR/Cas12a	Grain weight increased	Wang et al. (2021a)
	Oryza sativa	OsAAP3	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Tiller number increased	Lu et al. (2018)
	Oryza sativa	OsHXK1	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Chlorophyll content increased; photosynthesis improved. Hence, the grain number per panicle increased	Zheng et al. (2021)
	Oryza sativa	GS3	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Grain yield and quality improved	Huang et al. (2022a)
	Oryza sativa	OsSPL16	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Grain yield improved	Usman et al. (2020c)
	Oryza sativa	GS2	Agrobacterium-mediated transformation	Gain of function	CRISPR/Cas9	Grain yield, size, and length/width ratio increased	Wang et al. (2022a)
	Oryza sativa	OsGA20ox2	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	semi-dwarf phenotype observed. Compared to the wild type, yield per plant increased	Han et al. (2019)
	Oryza glumaepatula	GL9	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	The experiment demonstrated that the targeted gene positively regulates the wight and size and negatively regulates the chalkiness of the grain	Lin et al. (2023)
Size and shape	Cucumis sativus	CsTRM5: exons number 2 and 3 targeted	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Decreased length and increased diameter observed	Xie et al. (2023)
	Solanum lycopersicum	SlMYB3R3	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	elongated fruit shape observed	Zheng et al. (2022)
	Brassica rapa ssp. pekinensis	AGL19, AGL24	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Late-Bolting phenotype induced	Shin et al. (2022)
	Musa acuminate	MaGA20ox2	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	semi‐dwarf phenotype observed	Shao et al. (2020)
	Oryza sativa	OsPUB3	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	width and weight of the grain increased	Li et al. (2022b)
Nutritional components	Triticum aestivum L	TaSBEIIa	Vectors transferred into immature embryos of the plant using particle bombardment	Insertions and deletions	CRISPR/Cas9	The contents of amylose, protein, soluble pentosan, and resistant starch increased	Li et al. (2021a)
	Oryza sativa	OsGW2	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Grain with higher protein content induced; accumulation of important dietary minerals observed in the endosperm of grain	Achary and Reddy (2021)
	Oryza sativa	SBE genes	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Resistant starch content increased	Biswas et al. (2023)
	Solanum lycopersicum	5 genes in GABA shunt targeted	Agrobacterium-mediated transformation	Multi-site knock-outs	CRISPR/Cas9	The γ-aminobutyric acid content significantly increased	Li et al. (2018)
	Oryza sativa	OsVIT2	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Fe content in grain increased	Che et al. (2021)

Strigolactones (SLs) are classified as plant hormones that are known for impeding the process of tillering (shoot branching). CCD7 which stands for CAROTENOID CLEAVAGE DIOXYGENASE 7, is a gene responsible for the biosynthesis of SL in some plants like rice (Oryza sativa). In 2018, a group of scientists used the CRISPR-Cas9 system in Oryza sativa for selected mutagenesis of CCD7. OsCCD7 includes 7 exons by an ultimate production of a 609 amino acids protein. For the aim of targeting the intended gene to increase our knowledge of multiple functions of produced protein, they engineered two sgRNAs and named them sgRNA-1 and sgRNA-2 matched with the first and seventh exons respectively. After the regeneration of T0 plants, they found 22 transgenic plants with the formation of indels which shows the efficiency of the applied CRISPR-Cas9 system in the crops. The results of the experiment showed the phenotype changes in the studied plant like height reduction and tillering increase which could be a desired trait in agricultural biotechnology (Butt et al. 2018).

SPLs which stand for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE is a transcription factor family that plays an essential role in the development and growth of crops and plants (Preston and Hileman 2013). In 2019, Bao et al. employed assembled CRISPR-Cas9 with four sgRNAs in soybean (Glycine max) to induce mutation in four genes (GmSPL9, GmSPL9a, GmSPL9b, GmSPL9c) which encode SPL9 family in the intended plant to find out the genes function. The study’s results based on the examination of phenotypic traits of the studied plant indicate that the four mutated genes function as unessential transcription factors in the formation of Glycine max architecture (Bao et al. 2019).

Since food quality is directly engaged with human health, crop quality and its nutritional value have always been the major concern of consumers. Thus, for this application, a precise and easy-to-use gene editing technology like CRISPR-Cas to improve vitamins, fiber, proteins, bioactive compounds, and minerals in the food is needed. It also includes the modification of various crops' appearance, nutritional components, and other desired traits. CRISPR/Cas9 technology was used to optimize crop shape and size based on consumer preferences. Several genes/quantitative trait loci (QTLs) have been proposed as being responsible for crop appearance quality. Rice and tomatoes revealed the most information about crops and fruit shape and size regulation. The first QTL identified in regulating grain length, GS3 (GRAIN SIZE 3), has been successfully knocked out in five japonica rice varieties (Das et al. 2023). Many nutrient elements in vegetables and fruits have anti-inflammatory, anti-cancer, and antioxidant properties. Biofortification programs using CRISPR-Cas for diverse nutrients such as carotenoid, γ-aminobutyric acid, iron, and zinc content in various crops have been implemented.

Pigmentation

Most of the pigments that define plants colors are composed of chlorophylls, polyphenols, carotenoids, Betalains, and anthocyanin. The edible plants color has a strong influence on consumer’s choice. For example, in western countries, people usually prefer to consume red-colored tomatoes, while consumers in Asia, normally choose pink tomatoes to consume. Genes control color in plants, therefore, a precise and cost-effective gene editing tool guides agriculturists through targeted marketing and leads to sustainable agriculture (Liu et al. 2021a). Manipulation of fruit color can thus be accomplished by interrupting genes involved in the pigment production pathway using CRISPR/Cas9 (Table 4). MYB12, as a flavonoid biosynthesis pathway transcription factor, regulates flavonoid accumulation and the pink skin phenotype. Pink-fruited tomatoes have been successfully grown by knocking down SlMYB12. furthermore, by targeting PSY1 and ANT1, researchers were capable of producing yellow and purple tomatoes. DcMYB7, an R2R3-MYB, was knocked out in the solid purple carrot using CRISPR/Cas9, resulting in yellow roots (Negi et al. 2022).

Table 4.

Research studies using the CRISPR-Cas system focused on pigmentation

Plant species	Target	Type of manipulation	Type of CRISPR-Cas	Result	References
Gentiana scabra	Gt5GT, Gt3′GT	Knock-out	CRISPR-Cas9	The accumulation of delphinidin observed	Tasaki et al. (2019)
Brassica oleracea	MYBL2	Base editing	CRISPR-iSpyMacCas9	purple varieties developed from green varieties	Khusnutdinov et al. (2022)
Torenia fournieri	F3H	Gene suppression	CRISPR-Cas9	Pale blue flowers generated	Nishihara et al. (2018)
C. sinensis L. Osbeck	β-LCY2	Knock-out	CRISPR-Cas9	Lycopene and anthocyanin accumulated in the fruit	Salonia et al. (2022)
Daucus carota	F3H	Knock-out	CRISPR-Cas9	anthocyanin accumulation stopped	Klimek-Chodacka et al. (2018)
Gentiana scabra	GST1	Loss of function	CRISPR-Cas9	anthocyanin accumulation decreased and resulted in the generation of white and pale blue flowers	Tasaki et al. (2020)
Brassica oleracea var. alboglabra	BoaCRTISO	Loss of function	CRISPR-Cas9	Chlorophyll and Carotenoid concentration decreases and yellow color is resulted	Sun et al. (2020)
Solanum lycopersicum	SlSGR1	Knock-out	CRISPR-Cas9	More Carotenoid were obtained compare with wild type	Ma et al. (2022)
Solanum lycopersicum	PSY1, SGR1, MYB12	simultaneous knockouts	CRISPR-Cas9	The red-colored fruit was altered to green-colored fruit. Backcrossing the mutant generation with the wild generation can result in various colored fruit from brown to light-green	Yang et al. (2023)
Brassica napus	BnaC09.ZEP, BnaA09.ZEP	Knock-out	CRISPR-Cas9	Carotenoid profile changed and the color of the plant changed to orange from yellow	Liu et al. (2020a)

Former studies on the modification of ornamental flower color imply that the CRISPR/Cas systems can be used for research on plants’ functional analyses and pigmentation. For example, Masahiro and his colleagues tried to induce mutations using the CRISPR-Cas9 system in the F3H gene which stands for flavanone 3-hydroxylase in the torenia fournieri that has a chief responsibility in the biosynthesis of flavonoid. Mutation in the F3H gene was in charge of the white color in torenia Fournieri. Modification of flower color using CRISPR/Cas9 successfully achieved 80% in the studied plants which shows CRISPR-Cas9’s great potential using for intended gene mutation (Nishihara et al. 2018).

Introducing crops with less water demand and drought tolerance

Water demand is growing briskly more than two times of the human population. Additionally, agriculture is in fact the main water consumer on the planet and estimations say about 70% of the water use goes to this activity worldwide (Leonel and Tonetti 2021). Therefore, it is crucial to introduce new crops requiring less water and/or resistance against drought (Table 5). CRISPR-Cas technology can offer these characteristics to crops and plants. With the rapid advancement of CRISPR/Cas technology, The creation of a C4 plant model will speed up our understanding of the molecular processes underlying the Hatch-Slack pathway's ability to fix CO₂, and it may also make it possible to introduce C4 pathway genes into C3 plants, leading to the development of new crops with higher biomass yields and reduced nitrogen and water requirements to satisfy the world's increasing demand for bioenergy and food production (Bandyopadhyay et al. 2020; Peng and Zhang 2021).

Table 5.

CRISPR-Cas technology has been used in some experiments to introduce crops with lower water demands and drought tolerance, aiming to cut down on the amount of water used in agriculture

Crop species/ plant models	Target	Transfer method	Type of manipulation	Type of CRISPR-Cas	Result	References
Vitis vinifera	VvEPFL9-1	Agrobacterium-mediated transformation	Loss of function	CRISPR/Cas9	Intrinsic water-use efficiency enhanced	Clemens et al. (2022)
Solanum lycopersicum	SlARF4	Agrobacterium-mediated transformation	Loss of function	CRISPR/Cas9	Resistance of plant to water stress enhanced	Chen et al. (2021)
Triticum aestivum	TaSal1: exons 4,5, and 7 targeted	Transfer of vector by gene gun	Knock-out	multiplex sgRNA-CRISPR/Cas9	Water loss reduced	Abdallah et al. (2022)
Zea mays	ARGOS8	Agrobacterium-mediated transformation	Knock-in	CRISPR/Cas9	Higher yield under drought stress	Shi et al. (2017)
Oryza sativa	OsDST	Agrobacterium-mediated transformation	Loss of function	CRISPR/Cas9	Drought and salt tolerance increased	Santosh Kumar et al. (2020)
Glycine max	GmHdz4	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Drought tolerance increased	Zhong et al. (2022)
Oryza sativa	IPA1	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	The experiment demonstrates that the IPA1 gene plays a positive role in drought tolerance by activating SNAC1 gene	Chen et al. (2023)
Brachypodium distachyon	BdRFS	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Mutated plants showed decreased drought tolerance. Hence, the findings approve the positive role of BdRFS in drought stress	Ying et al. (2023)
Oryza sativa	OsLKP2	-	Knock-out	CRISPR/Cas9	Drought tolerance increased	Shim et al. (2023)
Oryza sativa	OsSAPK3	Vector inserted into protoplasts	Loss of function	CRISPR/Cas9	Mutated plants showed decreased drought tolerance. Hence, the findings approve the positive role of OsSAPK3 in drought stress	Lou et al. (2023)
Oryza sativa	OsDSR2	-	Knock-out	CRISPR/Cas9	Drought and salt tolerance increased	Luo et al. (2023)
Oryza sativa	OsNPF8.1	Agrobacterium-mediated transformation	Knock-out	CRISPR/Cas9	Mutated plants showed decreased drought and salt tolerance. Hence, the findings approve the positive role of OsSAPK3 in drought and salt stress	Diyang et al. (2023)

Most of the previous and recent studies on CRISPR applications trigger single genes knock-out and knock-in or create single base changes, but the progression of this technology enables and develops more applications such as rearrangement of chromosomes, say translocation, crossover, or inversion which can lead to amelioration of plant productivity or any other favorite traits. Altogether, we can comprehend that the CRISPR-Cas9 system, can give rise to our knowledge of various plants and crops genes related to beneficial traits and we can employ the data obtained by CRISPR in green biotechnology to reach sustainability (Rönspies et al. 2021).

CRISPR-Cas technology leads us through sustainable environment

The deleterious effects of climate change and pollution largely endanger our planet and all the different ecosystems on it. Climate change can lead to dramatic changes in temperature, rainfall patterns, distribution, etc. The concentration of Greenhouse gases (GHGs), mostly CO₂ has unfathomable effects on climate change, therefore, sustainable methods to capture and reduce CO₂ and reduce our carbon footprint are the most effective ways to confront with climate change crisis. Based on various research, the most sustainable approaches in terms of long-term environmental safety and economy are bio-based tactics. Accordingly, CO₂ bio-sequestration via organisms has arisen as a potential method of fixing CO₂ into biomass via the process of photosynthesis which in turn is an effective approach to producing value-added products and bioenergy (Singh and Dhar 2019). New genomic methodologies capable of producing genetically modified organisms using gene-splicing biotechnology techniques and recombinant DNA technology can lead to improvements in sustainable and renewable biofuel and biomaterial production from microorganisms (Lin et al. 2018; Shahcheraghi et al. 2022). Among molecular biology techniques, CRISPR-Cas technology offers an exceptional capacity for targeted genome editing, displaying a more accurate gene knock-out/knock-in system. The most recent advancements in CRISPR-Cas technology may open the door for the advancement of microbial biorefineries capable of producing potentially enhanced biofuel (Lin et al. 2018). By analyzing genetic processes to better microbial strains, this technology can help in the fight against greenhouse gas production (Asadian et al. 2022). Due to the short lifetime and fast-growing time of agricultural crops such as rice, a research group named Innovative Genomics Institute (IGI) founded by Jennifer Doudna, the co-inventor of CRISPR technology, proposed a novel method for bio-sequestration by optimizing traits of crops to capture and storage of CO₂ from the environment. Thus, this process slows down the harmful effects of climate change. Via this strategy, the photosynthesis and root systems of crops can be improved and a large amount of biochar and bio-oil could be produced. This project is funded by an 11-million-dollar grant by foundations that demonstrate the promising future of this technology and its parts in assuring the future of our blue planet (Crownhart 2022). CRISPR technology also can be exploited to mitigate carbon-based pollutants as the major origin of environmental pollution and has the ability to accelerate bio-based approaches for environmental remediation such as bioremediation. To sum it up, CRISPR technology offers us sustainable methods and approaches that guide us toward a sustainable environment (Yong et al. 2021).

CRISPR-Cas applications in biofuel production

Biofuel can be defined as a renewable source of energy and classified among cost-efficient and carbon–neutral technologies such as biogas, biohydrogen, biodiesel, biobutanol, syngas, and bioethanol (Zahed et al. 2021). Biofuels fall into four generations. from first to fourth, biofuels respectively derived from edible biomass, non-edible biomass, algal biomass, and genetically engineered organisms (Ganguly et al. 2021). Microorganisms, including microalgae and bacteria, represent a promising and sustainable avenue for biofuel production, offering a green alternative to conventional fossil fuels. This potential stems from their intricate metabolic pathways, encompassing processes such as fatty acid metabolism, the mevalonate pathway, and sugar catabolism. These pathways provide a foundation for efficient biofuel synthesis. The process of biofuel production driven by microorganisms comprises three essential phases: pretreatment, hydrolysis, and fermentation (Rodionova et al. 2017; Javed et al. 2019). Progressions in the process of biofuel production, are advantageous and beneficial due to its characteristics such as reduction in emission of greenhouse gases, cost-effective features and its part in the ongoing and future of the energy market; Thus, makes it an interesting field to study. Additionally, the application of forward-looking technologies for the improvement of biofuel production is vital, therefore, CRISPR-Cas systems as a sound molecular metabolic and genetic engineering tool can be applied for biofuel generation (Shanmugam et al. 2020).

There are several genes related to biofuel tolerance and production, inhibitor tolerance, thermostability, and enzymes associated with the process of biofuel (Table 6). All of those genes and proteins can be engineered via CRISPR-Cas machinery (Otoupal and Chatterjee 2018; Javed et al. 2019). For instance, modification in cAMP Receptor Protein increases oxidative stress and biofuel tolerance in Escherichia coli (Basak and Jiang 2012; Chong et al. 2013; Javed et al. 2019). Microbes’ metabolic pathways of biofuel production can be affected by some dosage of the end product in their culture because of the antimicrobial activities of the final products. by genome engineering of genes related to the biofuel tolerance, their tolerance against the end product can be induced (Otoupal and Chatterjee 2018; Javed et al. 2019) Modifications in RMD6, MSN2, and SSK2 in the Saccharomyces cerevisiae can increase the bacterial toleration against ethanol and furfural. Additionally, modifications in the gene of SSK2 via CRISPR-Cas9 can ameliorate the production of biofuels. Studies report that gene Cel7A in the Trichoderma reesei deteriorates product inhibition of enzymes related to biofuel production processes (Cho et al. 2011; Park et al. 2012). based on former studies, CRISPR-Cas systems can promote microorganisms’ applications say, biofuel production which may lead to a greener environment (Feng et al. 2020).

Table 6.

genes related to biofuel production in bacteria, plant, algae, and fungus which have been targeted via CRISPR-Cas technology to produce biofuels

Strain	Target(s)	CRISPR-Cas System	Type of manipulation	Type of fermentation	Medium	Result	References
Bacillus subtilis (bacteria)	nprB, vpr	CRISPR-Cas9	nprB and vpr deleted via NHEJ	Shake Flask Fermentation	LB medium	2.4 times more isobutanol produced than in the wild type	Tian et al. (2022)
E. coli SSK42 (bacteria)	adhE	CRISPR/Cas9	adhE knocked-out and synthetic butanol pathway construct combined into the genome	fed-batch cultivation	xylose in minimal media	The butanol and ethanol titer respectively reached up to 4.32 g/L, and 8.5 g/L of total solvent	Abdelaal et al. (2019)
Nicotiana tabacum L. (plant)	NtFAD2-2	CRISPR/Cas9	Knock-out	-	-	Oleic acid content was increased up to 67% which makes it more capable of biodiesel production	Tian et al. (2020)
S. cerevisiae SCGFA (Fungus)	GPD2, FPS1, ADH2	CRISPR/Cas9	Multiple gene knock-outs	batch fermentation	YPD medium	1.18 times more bioethanol produced than in the wild type	Yang et al. (2022)
Clostridium acetobutylicum DSM 792 (bacteria)	bdhA, bdhB, bdhC	CRISPR/Cas9	Knock-out	batch fermentation	Modified Gapes medium	ethanol and butanol production decreased which demonstrated the role of targeted genes	Wasels et al. (2020)
Tetraselmis sp. (green microalga)	AGP	CRISPR/Cas9	Knock-out	bubble column photobioreactor	artificial seawater	3.1 times more lipid content was produced than in the wild type	Chang et al. (2020)
Bacillus licheniformis (bacteria)	pgsBCAE operon, sacB	CRISPR/Cas9	Knock-out	fed-batch fermentation	corn steep liquor	mucus synthesis blocked and 2, 3-butanediol bioproduction enhanced	Song et al. (2021)
Camelina sativa (plant)	CsFAD2	CRISPR/Cas9	Knock-out	-	-	The content of Monounsaturated Fatty Acid increased up to 57.8%	Lee et al. (2021a)
Kluyveromyces marxianus (fungus)	KmARO10 combined into URA3 locus	CRISPR/Cas9	knock-in	fed-batch fermentation	YPD medium	2-phenylethanol and 2-phenylethyl acetate production, respectively, improved up to 10% and 146%	Li et al. (2021b)
Chlamydomonas reinhardtii (algae)	PLA2	CRISPR/Cas9	Knock-out	rotary shaking incubator under continuous white light	Tris–acetate phosphate	Lipid production improved up to 64.25%	Shin et al. (2019)

CRISPR-Cas in bioremediation

Bioremediation is a cost-effective, non-toxic, and eco-friendly biotechnology process in which environmental pollution is remediated via microbial pathways like enzymatic pathways in different environments such as terrestrial or aquatic (Dangi et al. 2019; Sharma and Shukla 2022). Disposal of substances containing hydrocarbons like pesticides, plastics, fertilizers, etc., or any other detrimental chemical compounds is vital for the environment because these compounds may lead to bioaccumulation or bio-magnification which endanger ecosystems’ safety (Azubuike et al. 2016; Sharma et al. 2018). Bioremediation mostly relies on the enzymatic activities of microorganisms besides some effective environmental factors such as pH, salinity, nutrient, or temperature of the studied environment. There are many microorganisms that produce enzymes that are beneficial for environmental bioremediation such as Phosphotriesterases, Peroxidases, Oxygenases, Lipases, Laccases, Carboxylesterases, Haloalkane dehalogenases, Cellulases, and many other enzymes (Zahed et al. 2022). Genetic engineering methods such as CRISPR-Cas9 technology give experts in synthetic biology an appropriate approach to upsurge the enzymes production or other microbial production with assistance the hazardous compounds degradation via transferring the coded genes of enzymes into another microbial host with anticipated characteristics to overproduce recombinant strains with more bioremediation capability (Kumari and Chaudhary 2020; Jaiswal and Shukla 2020). The organisms engineered via the CRISPR-Cas system also can be implied as more efficient than before for water and wastewater treatment to reuse the water and wastewater for crop irrigation and/or other activities that require water consumption (Leonel and Tonetti 2021).

Two approaches after identifying the target genes can be used to improve microorganisms' capacity for bioremediation through genetic engineering: 1- enhancing the degradability and strain tolerance of contaminant, such as hydrocarbons, heavy metals, etc., or 2- expressing and synthesizing compounds that assist in degradation, like biosurfactants or antifouling components (Panigrahi et al. 2019; Zahed et al. 2022).

Phytoremediation

The phrase phytoremediation refers to the cleanup of contaminants using plants (Sarma et al. 2021). It is essentially a strategy for restoring soil fertility and cleansing the soil. With the use of CRISPR technology, it is possible to modify DNA, which may subsequently be used to alter the gene sequencing of plants and lessen the negative impacts of heavy metals and other contaminants in the soil. Many model phytoremediators have had their genomes entirely or partly sequenced, including Noccaea caerulescens (a hyperaccumulator of Cd, Zn, and Ni), Arabidopsis halleri (hyperaccumulator of Cd and Zn), Hirschfeldia incana (capable of withstanding Pb), Pteris vittata, and Brassica juncea, as well as several other species (Saxena et al. 2020; Agarwal and Rani 2022).

The NAS1 gene, which codes for the enzyme Nicotianamine synthase, has been added to Arabidopsis and tobacco plants. These plants have demonstrated enhanced tolerance to metals like Cd, Cu, Fe, Mn, Ni, and Zn as well as higher Mn and Ni uptake. In poplar, tobacco, and Arabidopsis plants, overexpression of metallothionein-encoding genes (MTA1, MT1, and MT2) enhanced Cd, Cu, and Zn resistance and accumulation (Basharat et al. 2018; Patel et al. 2022). It is known that the expression of the metallothionein gene, MT2b, together with the up- and down-regulation of genes involved in the generation and catalysis of abscisic acid, increases Hirschfeldia incana’s capacity to tolerate and accumulate Pb (Auguy et al. 2016). In Brassica juncea plants, Se tolerance and accumulation were enhanced by the transfer of the genes APS and SMT, which are in charge of producing ATP sulfurylase and selenocysteine methyltransferase, respectively (Basharat et al. 2018; Patel et al. 2022).

CRISPR-Cas applications in biosurfactant production

Biosurfactants (BS) as secondary and surface-active metabolites are eco-friendly, biodegradable, and non-noxious amphiphilic molecules synthesized by some microorganisms and plants with various applications in bioremediation, food, agriculture, medicine, pharmaceutics, biofilm, wastewater treatment, cosmetics, etc. (Fig. 3) (Hajfarajollah et al. 2018; Markande et al. 2021). Also, some types of BSs show antimicrobial activities, therefore, these biological compounds also can be used for the biocontrol of pathogenic microbes in plants and crops. The BS producers can thrive on many cheap substrates say biochar, agricultural waste, carbohydrates, plant oils, etc. (John et al. 2021). The most common types of BSs include glycolipids, lipopeptides, fatty acids, polymeric surfactants, phospholipids, and neutral lipids (Jimoh and Lin 2019). BSs are biological equivalents of chemical surfactants. they are more environmentally friendly and greener than chemical surfactants but are not economically reasonable to be widely used in different industries. Despite BSs’ advantages, their production cost is still around twelve times higher than chemical surfactants (de Oliveira Schmidt et al. 2021). For overcoming this problem, one of the best and most effective solutions is the genetic engineering of BS producer microorganisms with effective molecular tools such as the CRISPR-Cas9 system (Jimoh et al. 2021; Zahed et al. 2022).

Fig. 3.

Biosurfactant has various applications in many fields that help the planet and individuals to become more sustainable and healthier. CRISPR-Cas9 genome editing technology can introduce microbial strains with the production ability of cost-effective biosurfactants

There are many genes involved in the production of BSs in microorganisms, for example, rhlA, rhlB, rhlR, and rhll in Pseudomonas aeruginosa are responsible for the production of rhamnolipid BS or gene ituD in Bacillus subtilis RB14 has a chief role in production of lipopeptide BS or LuxR-type transcriptional regulators in strain SBW25 of Pseudomonas fluorescens have a significant part in the production of a lipopeptide BS (Markande et al. 2021).

Studies demonstrate that BS production by genetically engineered microorganisms, increases the bioremediation rate of various environmental contaminants say, petroleum hydrocarbons. In the case of bioremediation of contaminants with the help of BS, the more genetically engineered microbes produce BS, the surface tension reduces more rapidly and after reaching the Critical Micelle Concentration (CMC), the BS’s monomers start to form micelles. This phenomenon increases the bioavailability of contaminants and ameliorates the degradation rate of pollutants from the environment (Ali Khan et al. 2017). Furthermore, statistics say that approximately 70 to 75% of prevailing chemical surfactants are produced from petroleum, therefore broad use of BS reduces carbon footprint (Lee et al. 2018). Hence, the use of CRISPR-Cas9 in BS-producing microorganisms with the purpose of producing genetically engineered microbes with higher potential of BS production leads to phenomena like carbon footprint reduction, biocontrol of crops, and more effective bioremediation methods which result in a cleaner and more sustainable environment and agriculture (Nikolova and Gutierrez 2021; Goswami and Deka 2021; Zahed et al. 2021).

Conclusion

As a biotechnological tool, CRISPR-Cas technology can be utilized to combat a number of the crises we are currently facing, including environmental pollution, climate change, etc. The current review, which is based on more than 200 scientific articles and studies, concludes that CRISPR-Cas can hasten the transition to a sustainable world via the available applications in green biotechnology. In the agricultural field, this technology via genetic engineering and amelioration of crops’ traits such as disease resistance, herbicide resistance, yield, nutritional value, pigmentation, and water demand, takes us a step forward to sustainability in agriculture. Additionally, this technique enables us to extend the domestication of plants, advancing the cause of sustainable agriculture. Using CRISPR-Cas technology, it is also possible to modify plants and microbes to enhance carbon capture and reduction through bio-sequestration, bioremediation, phytoremediation, and production of value-added products such as biochar, biosurfactant, and biofuel. Enhancing carbon capture and reduction techniques expedites GHG emissions reduction and aids in our fight against a destructive phenomenon like climate change. The aforementioned technology can speed up bio-based remediation of contaminants, which is effective and environmentally benign. Bio-based products can also lessen carbon footprint. As a result, CRISPR-Cas technology holds out hope for a path toward sustainable agriculture and environment as well as for helping us combat environmental disasters.

Funding

There is no source of funding to declare.

Data availability

All of the datasets generated and/or analyzed during the current review study listed in the “References” section of this article.

Declarations

Conflict of interest

The authors declare that they have no competing interests. There is no human participants and/or animals involved in this research.