**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 doublestrand 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 mitochondrial and chloroplast genomes. Despite the identification of several meganucleases, it 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 merging 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 phytopathogenic 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 populations 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
