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3. In the near future: the importance of genome editing in herbs and the methodologies
Genome editing technologies have potential for nutrition due to climate change, reduced agricultural fields, and increased plant stressors. New global agriculture and food production strategies indicate that the revision of the food genome has been important. Actually, the history of genome editing was established over the 1980s as plant breeding. This innovation has supported both nutrition and the food and pharmaceutical industries.
Genome editing technology is a type of genetic engineering in which DNA is inserted, suppressed, altered, or replaced in the genome of a living organism.
Genetic material is randomly inserted into the genome of a host by focusing on specific locations. However, it has been reported that random insertion of DNA into the host genome is a disadvantage of this technology because of disruption or alteration of other genes in the organism [20]. So, there was a concern about genetically modified products. Nevertheless, in the 2000s, genome editing has been successfully accomplished for both animal and plant systems with the use of artificial or natural region-specific nucleases and genome editing technologies. Genome editing technology has become a powerful method for functional genomics and crop selection studies in comparison with the randomized method [21].
Some plant transformation techniques are used for genome editing;
administration of polyethylene glycol in protoplasts [22], microparticle bombardment [23], WHISKERS™ [24], and Agrobacterium [25]. They can deliver these genome editing reagents to plant cells [26, 27]. More recently, genome editing methods have started to be used to improve our understanding of plant gene functions and the alteration and enhancement of plant genes. The genome editing allows the addition, removal, or modification of the desired genes in the genome by creating double- strand breaks (DSBs) with the specific nucleases of the region. There are four ways to accomplish this: 1. Meganucleases; 2. Zinc finger nucleases (ZFNs); 3. Transcription activator-like effector nucleases (TALENs) and 4. Clustered regularly interspaced short palindromic repeats (CRISPR).
Meganucleases are regarded as the most specific naturally occurring restriction enzymes that are also mobile genetic compounds. They are synthesized in mitochon- drial and chloroplast genomes. Despite the identification of several meganucleases, it
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is naturally impossible to find a suitable enzyme for each region. A new enzyme model is needed for each study. Meganucleases have been successfully used to target DNA insertions in various plants, such as maize, tobacco, and Arabidopsis species [28].
Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by merg- ing a DNA-binding domain from a zinc finger to a DNA cleavage domain. ZFNs are synthetic nucleases, which were first discovered in TFIIIA from Xenopus laevis frogs.
The zinc finger protein motif encounters transcribing factors and recognizes the DNA sequences. In fact, ZFNs can be designed to bind and divide any of the DNA sequences. However, for each region of the genome, ZFN must be regenerated. This increases costs and is time-consuming. Several studies have been carried out on plant genome modification using ZFNs, in particular Arabidopsis plants, tobacco, maize, and soybeans [29–32]. Additionally, genome editing in microalgae with ZFNs was first reported for Chlamydomonas reinhardtii, which was the adapted model strain [33, 34].
Transcription activator-like effector nucleases (TALENs) are similar to those in ZFN. The targeting strategy is delivered by linking pairs to two tightly spaced DNA sequences in both systems. The TALEN proteins were obtained from the phytopatho- genic bacterium Xanthomonas. The TALENs method was considered to be successful for Arabidopsis, rice, tobacco, barley, Brachypodium, and corn [35]. Nonetheless, the microalgae Phaeodactylum tricornutum was highlighted by the TALENs method to improve lipid accumulation [36]. It was also reported that genome editing in P. tricornutum and Chlamydomonas has been established by TALENs [37, 38].
Clustered regularly interspaced short palindromic repeat (CRISPR) is the most important method for plant biotechnology whose working principle is based on RNA-mediated nucleases [39]. This is a system discovered from Streptococcus pygonese and named CRISPR/Cas9 system. Clustered regularly interspaced short palindromic repeats (CRISPRs)-case-mediated immunity in bacteria provides bacterial popula- tions with protection from pathogens. However, they are also exposed to the dangers of autoimmunity by developing protection that targets their own genomes. CRISPR/
Cas vectors have a replication origin and marker gene and also have a power promotor with Cas genes. This makes it possible to target several genes, and consequently, this technology costs less than others. The scientists reported that the Cas9 system could be used to modify the human genome as well as the plant genome [39, 40]. There are two main strategies: using RNA as vectors or transferring a functional nuclease directly into the cells of plants.
Besides its applicability in plant biology, the main focus of CRISPR/Cas system is producing heritable mutations within NHEJ-mediated (NHEJ: nonhomologous end joining) in many species. Also, it’s possible to add a DNA fragment via HDR (HDR:
homology directed recombination) to a desired region in the plant genome with the CRISPR system; however, a few number of studies have been conducted [41, 42].
Several plant genomes have been modified with CRISPR technology: rice, wheat, corn, tomato, potatoes, cucumber, orange, soybean, tobacco, lemon, and microalgae [32, 43]. The studies provided comparative data, including mutagenesis, efficiency, truncation specifications, potential for generating chromosomal deletions, or adding CRISPR genes [39, 44]. Also, it was reported that there have been several studies;
nontoxicity mutation with mediated-CRISPR such as microalgae [45], basic biological studies such as on the opium poppy [46], and improving the quality of products such as tomatoes [47]. The CRISPR system is a multiplex engineering of the genome, which means that multiple genes may be targeted.
In addition, the main advantage of the CRISPR system is that it prevents the gene from moving between organisms and problems related to gene transporting. Also, no
difference occurs in the next generation of organism, biallelic may be provided, and heterozygous and homozygous mutations may be generated [48]. Svitashev et al. [49]
and Woo et al. [50] have conducted studies with lettuce, rice, and corn to get success- ful mutations and modified fields with no alien DNA and marker. In addition, some microalgae, namely C. reinhardtii, Chlamydomonas, and P. tricornutum, have been edited successfully without cytotoxic effects [45, 51, 52]. According to the literature, genome editing with CRISPR/Cas engineering for single nucleotide resolution edit- ing, multiple gene editing, transcriptional regulation, and genome-wide modifica- tions of Saccharomyces cerevisiae have been shown [53, 54]. S. cerevisiae is an important eukaryotic yeast for the biosynthesis and biofuels [55, 56]. However, there are still some limitations and challenges, in particular the application of CRISPR could limit the effectiveness of yeast processing.
All these technologies have been reported for improving plant micronutrients, such as flavonoids, phenols, saponins, tannins, etc. [57]. These are bioactive com- pounds known as medicines that are important to health. In particular, the CRISPR/
Plants Methods Improved trait
Rice TALENs Increased fragrance content
Rice CRISPR/Cas9 Functional metabolites (amylose, Proanthocyanidins, anthocyanidins, beta carotene)
Wheat CRISPR/Cas9 Increased protein (reduce gliadins)
Corn ZFNs Antinutrient (reduce the phytic acid content) Corn TALEN Antinutrient (reduce the phytic acid content) Corn CRISPR/Cas9 Protein (reduce zein protein)
Potato TALEN Reduced browning, antinutrient (reduce steroidal glycoalkaloids) Toxic substance (reduce sugar and acrylamide)
Potato CRISPR/Cas9 Reduced browning, starch (amylose), anti-nutrient (absence of steroidal glycoalkaloids)
Oilseed CRISPR/Cas9
TALEN
Reduced oil content,
Reduced polyunsaturated fatty acids, Tobacco Meganuclease Reduced nicotine levels
Tomato TALEN Functional metabolite (increased anthocyanin)
Tomato CRISPR/Cas9 Functional metabolite (increased anthocyanin, aminobutyric acid content)
Tomato ZFN Antinutrient (reduced anti-nutrient oxalic acid) Grape CRISPR/Cas9 Antinutrient (reduced tartaric acid level) Sage (Salvia
miltiorrhiza)
CRISPR/Cas9 Decreased phenolic acid contents
Pomegranate CRISPR/Cas9 Changes the galloyl-glucose conjugates Grapevine CRISPR/Cas9 Lack of pigments phenotypes
Papaver CRISPR/Cas9 Biosynthesis flux of morphine, thebaine, etc.
Banana CRISPR/Cas9 Functional metabolites (Beta carotene)
Table 1.
Some nutritional quality-improved foods by gene-editing technologies (Prepared according to literatures; Ku and Ha [64], Scarano et al. [65], Dey et al. [66]).
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Cas9 technology can be used to target genes in medicinal plants and their compounds.
In addition, this technology can tolerate environmental stress along with quality and performance. There have been successful studies about plant biosynthetic pathways with CRISPR/Cas9, such as tomato for gamma-aminobutyric acid, banana, and rice for beta carotene [58–61]. Also, CRISPR/Cas9 is an effectible technology for bacte- rial resistance of herbs and plant-derived products and against climate changes
[62, 63]. Genome editing technology involves more controlled mutations, and genetic improvement is less time-consuming.
Biotechnology approaches have been interpreted in the context of genome editing technologies over the years. Secondary plant metabolites that belong to genome tech- nology are pharmacologically important as well as nutritional (Table 1). However, the editing of the genome is still in its beginnings in plants and their contents. As new and interesting results are obtained in this field, new technologies will emerge.
4. The differences between functional foods and herbs with edited