Keywords

Introduction

For centuries, medicinal plants have been used by us for the production of a plethora of unique metabolites (Calabrò, 2015). Metabolites are biomolecules which formed during the metabolism of an organism. Metabolites have crucial roles in intracellular and intercellular signalling pathways, growth, development and defence against pathogens. Moreover, some metabolites are economically important for the production of drugs, flavourings, pesticides, aromatic compounds etc. (Hussain et al., 2012). There has been evidence that secondary metabolites of plants could serve as potential pharmaceutical leads for reducing human deaths (Dey et al., 2017b). Various natural compounds have been implicated in the reversibility of drug resistance (Das et al., 2021; Guo et al., 2017; Wang et al., 2015). The efficacy of secondary metabolites in medicinal herbalism has also been demonstrated by extensive clinical and preclinical studies (Anand et al., 2020; Kaur et al., 2020a, 2020b). For enhancing the production of valuable plant secondary metabolites, several biotechnological interventions have been applied (Dey et al., 2017a).

Plants produce different secondary metabolites that are restricted to specific groups of plants within the plant kingdom. In recent times, the world has shifted towards natural products due to their significant role in medicine without severe side effects. For that, evolving demand for secondary metabolites resulted in boundless interest mainly in the possibility of changing the production of bioactive plant metabolites via targeted metabolic engineering, elicitation, endophytes, bioreactors, precursor feeding, nanotechnology and other specialised biotechnological approaches (Calabrò, 2015; Hussain et al., 2012; Narayani & Srivastava, 2017; Verpoorte & Memelink, 2002). Metabolic engineering can be applied in several ways to increase metabolite yields, including by blocking catabolism, reducing competitive pathways and disabling rate-limiting steps (Verpoorte et al., 1999). Modulations in the production of plant metabolites can be accomplished by targeting regulatory genes for their biosynthetic pathways. Enhancement of secondary metabolite production can sometimes be achieved by targeting a single pathway gene that can act as a transcription factor involved in regulating multiple pathway genes or through knocking out the gene which is responsible for the suppression of natural product production (Staniek et al., 2013; Verpoorte & Memelink, 2002).

There is an increasing demand for genetic engineering strategies; however, conventional genetic engineering approaches have numerous shortcomings, such as being unable to manipulate the genomes of large plants. A variety of tools for genome editing at the transcriptional level such as zinc-finger nucleases (ZFNs), meganucleases (MNs), transcription activator-like endonucleases (TALENs) and CRISPR are helping to resolve the difficulties of specific genome editing of plants (Rehman et al., 2021; Chen & Gao, 2015). ZFNs and TALENs initiate genome editing by employing Fok-I, a protein-based endonuclease for gene knockout through producing double-stranded breaks (DSBs) in the DNA target which further arouse error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) at precise genomic positions (Gaj et al., 2013; Khan, 2019). However, in order to utilise the TALENs and ZFNs techniques, two distinct protein hybrid designs are required which can identify sparsely existing DNA flanks (Li et al., 2011). Furthermore, these techniques are lengthy and less precise, which prompted scientists to develop an efficient genome editing method. Successful gene manipulation of plants has been achieved with CRISPR and associated proteins, for example, CRISPR-associated protein Cas9 (Upadhyay & Sharma, 2014). Figure 8.1 presents the key steps of genome editing through the CRISPR tool in plants.

Fig. 8.1
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The key steps of genome editing through the CRISPR technique in plants

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Background Study of CRISPR-Cas9

The CRISPR/Cas is part of the adaptive immune system of bacteria and a few archaea. Foreign particles like viruses and plasmids are repelled by the CRISPR/Cas mechanism. The foreign genetic material (DNA) will be sliced and reorganised and its genetic sequence preserved for when the same virus or plasmid invades again, the genetic material will be recognised (Koonin & Makarova, 2013; Koonin & Makarova, 2009). CRISPR/Cas system contains sgRNA (single guide RNA) and Cas9 endonuclease which form a complex that can cleave target DNA sites. CRISPR-Cas9 genome editing system is functioning by introducing DSBs (Jinek et al., 2012). Previous gene manipulating nuclease platforms such as ZFNs and TALENS, could initiate DNA DSBs; however CRISPR-Cas9 differs from ZFNs and TALENS as Cas9 nucleases form a complex with the sgRNAs that guide specific DNA sequences within host nuclei and initiate DSB induction (Jinek et al., 2012). The most likely outcome of a DSB is the initiation of NHEJ mechanisms producing various mutations such as indel and substitutions. By introducing frameshift mutations, NHEJ mechanisms can be used in breeding, mutant library construction and high-throughput mutational screening (Barakate & Stephens, 2016). DSB formation can also initiate HDR, although that is less likely. The CRISPR/Cas technology is a new, popular and powerful gene editing tool and has wide application in gene manipulation in the field of genomic research due to its cost-effectiveness and efficiency (Cui et al., 2018). There are numerous uses of CRISPR/Cas9 in genetically modifying both medicinal and food plants and model organisms (Alagoz et al., 2016; Malnoy et al., 2016; Ricroch et al., 2017; Shan et al., 2014; Singh et al., 2020). CRISPR/Cas9 has successfully edited approximately 32 different plants, but the application of this technology to medicinal plants is still limited. Therefore, this chapter intends to summarise data available in the past pertaining to ten medicinal plant genomes edited with CRISPR. CRISPR-Cas9 toolkit limitations and prospective are also discussed in this chapter.

Secondary Metabolite Engineering of Plants with CRISPR/Cas9

It is believed that there are over lakhs of secondary metabolites, which are classified by structure, function and biosynthesis. Secondary metabolites are divided into alkaloids, terpenoids and steroids, fatty acid-derived compounds, polyketides and nonribosomal polypeptides and enzyme cofactors (McMurry, 2015). CRISPR/Cas offers an alternative production strategy of commercially important plant-derived natural products by modulating the phytochemical profile of medicinal plants with CRISPR/Cas9 technology. Table 8.1 presents application of gene editing with CRISPR/Cas in medicinal plants for the production of valuable secondary metabolites. In Fig. 8.2 we have highlighted different plant metabolites and their genome editing by CRISPR/Cas9 action.

Table 8.1 Gene editing with CRISPR/Cas in medicinal plants for the production of valuable secondary metabolites
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Fig. 8.2
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Plant metabolites and their genome editing by CRISPR/Cas9

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Atropa belladonna L.

Atropa belladonna L., alternatively referred to as deadly nightshade, is used extensively against anti-inflammatory and neuropharmacological disorders. Hyoscyamine is the major tropane alkaloid compound produced by A. belladonna which has activity against arrhythmias and organophosphate poisoning. This plant also produces the derivatives of hyoscyamine such as anisodamine and scopolamine (Hashimoto, 1992). Therefore, it is necessary to separate one compound from the others. However, due to their similar structures, this can be difficult to isolate compounds autonomously. So, it is crucial to develop A. belladonna without anisodamine and scopolamine, in order to achieve low costs for hyoscyamine separation. Additionally, hyoscyamine content in A. belladonna is very low and commercial demand for this compound is high; therefore, Zeng et al. (2021) develop high yields of hyoscyamine from A. belladonna plants for the first time, using CRISPR/Cas9 tool. The editing method, CRISPR/Cas9, disrupted the hyoscyamine 6β-hydroxylase (AbH6H) gene, initiated homozygous mutations in AbH6H and led to elevated hyoscyamine production, as well as decreased hyoscyamine 6β-hydroxylase function without anisodamine or scopolamine (Zeng et al., 2021). The editing method, CRISPR/Cas9, disrupted the hyoscyamine gene, initiated homozygous mutations in AbH6H and led to elevated hyoscyamine production, as well as reduced gene function. Hyoscyamine 6β-hydroxylase catalyses the 6β-hydroxylation of hyoscyamine which further produces anisodamine and subsequently converts it to scopolamine (Hashimoto & Yamada, 1986).

Brassica napus L.

Rapeseed oil ((B. napus) contains three major unsaturated fatty acids, including linoleic acid (18:2), linolenic acid (18:3) and oleic acid (18:1), as well as palmitic acid (16:0) and stearic acid (18:0), which are of high nutritional value (Nesi et al., 2008; Peng et al., 2010). Higher oleic acid content enhances thermal stability of the oil. Oleic acid content of napus is regulated by FAD2 (fatty acid desaturase 2 gene) loci. Standardised CRISPR/Cas9 technology has generated mutated BnaA.FAD2 through Agrobacterium-mediated transformation in B. napus. In an attempt to boost the content of oleic acid in B. napus genes using the CRISPR/Cas9 genome editing tool, mutated BnaFAD2 copies were successfully introduced. Their result suggested that modification by CRISPR/Cas9 system showed significant increase in the content of oleic acid by 80% and a notable decrease in linoleic acid and linolenic acid (Huang et al., 2020). The report also suggested that genome editing of polyploidy species is quite feasible and does not lead to chimeric modifications. One study using CRISPR/Cas9 technology to drive knockout mutations in FAD2 genes in B. napus resulted in an increase in the oleic acid (73%–80%) and reduction in linoleic acid (16%–9%). Though, 4-bp deletion of mutant fad2_Aa allele exhibited normal plant growth. Thus, knocking out via CRISPR/Cas9 technology can also be used to produce mutant plants that are agronomically more advantageous, such as mutant plants that contain higher oleic acid content important (Okuzaki et al., 2018).

Camelina sativa (L.) Crantz

CRISPR/Cas9-based gene editing was effective in enhancing the levels of oleic acid in the hexaploid C. sativa plant. Knockout of the FAD2 genes successfully increased seed oil. A significant increase in oleic acid content (16 % to over 50%) was observed, accompanied by a drop in linoleic acid (~16% to <4%) and linolenic acid (~35% to <10%). gRNAs targeted all three homologous FAD 2 genes simultaneously in Camelina seeds of T3 and T4 generations (Jiang et al., 2017). An alternative study used CRISPR/Cas9 genome editing technology to improve oleic acid content (from 10 to 62%) and reduce PUFA in the C. sativa seed oil using three closely related FAD 2 genes through targeted mutagenesis. The results of evaluating the mutations in three isologous CsFAD 2 genes over four generations have confirmed a large heritability of mutations (Morineau et al., 2017). In another experiment in allohexaploid C. sativa, CRISPR/Cas9 employed knockout mutagenesis by ethyl methanesulfonate which formed fatty acid elongase 1 (FAE1) gene mutants that resulted in the reduction in the C20–C24 very long-chain fatty acids (over 60%). Therefore, the inactivation of FAE1 genes enabled the improvement of oleic acid content or α-linolenic acid in seed oil. Interestingly, knocking out mutant fae1 showed normal growth and seed development (Ozseyhan et al., 2018). Others altered three conserved homologous genes (CsDGAT1 or CsPDAT1) via sgRNA to manipulate TAG (triacylglycerol) composition in the seeds of C. sativa. In addition, the seed of both CsDGAT1 and CsPDAT1 targeted lines shows wrinkled and shrinking surface (Aznar-Moreno & Durrett, 2017).

Dendrobium officinale Kimura and Migo

One of the largest groups of Angiosperm monocotyledon family, Orchidaceae, finds its uses not only in cosmetics, perfumes and for decoration purposes but also as natural remedies for the treatment of numerous diseases (Sut et al., 2017). The Chinese medicinal herb Dendrobium officinale, a species belonging to this family, produces many useful primary and secondary metabolites. There is also evidence to suggest that it heals yin deficiency disorders (Guo et al., 2020; Tang et al., 2017). Major bioactive compounds of D. officinale are polysaccharides, besides other compounds such as alkaloids, bibenzyls, essential oils, phenanthrenes and glycosides which have anticancer, antibacterial, antioxidant, anti-inflammatory, antidiabetic, antiviral, anti-ageing and hair growth-promoting properties (Tang et al., 2017; Teixeira et al., 2015). Metabolic profiling of D. officinale has led to the identification of several pharmaceutically active ingredients (Jin et al., 2016). The protocol for genome editing through CRISPR/Cas9 is well established for this orchid (Kui et al., 2017). This has not only eased the process of precise genetic manipulation of this plant but also helped in eliminating the controversies associated with transgenic acceptability. This knockout system reached 10–100% editing efficiency by using highly effective promoter (Cauliflower mosaic virus 35S), reporter genes β-glucuronidase (GUS), superfolder green fluorescence protein (SG) and vector (pCambia-1301-35SN) by using Agrobacterium-mediated transformation system. Five lignocellulose biosynthesis pathway genes, C3H (coumarate 3-hydroxylase), C4H (cinnamate 4-hydroxylase), 4CL (4-coumarate), CCR (cinnamoyl coenzyme A reductase) and IRX (irregular xylem5), were used to determine the efficiency of genome editing with CRISPR/Cas9 in D. officinale. The high efficiency of genome editing marks the entry of D. officinale in the new age of reverse genetics to decipher orchid gene functions which will further lead to the sustainable exploitation of this plant for our benefit (Kui et al., 2017).

Dioscorea zingiberensis C. H. Wright

Dioscorea spp. are medicinally important species of plants that are used for the extraction of diosgenin. The rhizomes of this species are used for the isolation of steroidal saponin, i.e. diosgenin, which has therapeutic significance (Pandey et al., 2017). Syombua et al. (2021), for the first time, evaluated the efficiency of CRISPR/Cas9 genome editing tool by targeting the phytoene desaturase gene (DrPDS) of Dioscorea alata. PDS (phytoene desaturase) catalyses the conversion of phytoene into both phytofluene and f-carotene (carotenoid precursors) (Mann et al., 1994). Results of this study proved that CRISPR/Cas9 efficiently induced site-specific disruption of the PDS gene with 83.3% editing efficiency and also induced phenotypical changes in yam. It is expected that the established CRISPR/Cas9 system in this species will serve as basal information for establishing editing protocols in other Dioscorea species. This will assist in functional genomics studies’ trait improvement in other related species like D. alata. D. zingiberensis (a perennial vine, commonly known as ‘yellow ginger’) is extensively used in Chinese medicines (Zhang et al., 2018). A range of biological effects like anti-inflammatory, anthelmintic, anti-thrombosis, cardiovascular, hyperlipidaemia and neuroprotection activity have been confirmed. In order to target the farnesyl pyrophosphate synthase gene (Dzfps) (the gene controlling diosgenin biosynthesis), the CRISPR/Cas9 system was used via an Agrobacterium tumefaciens-facilitated transfection method in D. zingiberensis. Farnesyl pyrophosphate (FPP) synthase is important for the production of isoprenoids such as carotenoids, ubiquinones, sterols etc. (Dhar et al., 2013). Cas9 and sgRNA expression cassettes are controlled by the OsU3 and 35S promoters, respectively, which are designed to target the Dzfps gene. Dzfps gene is involved in sequentially catalysing dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP) to produce FPP and finally squalene which is the precursor of diosgenin. Both Dzfps transcript level and squalene content were relatively decreased in mutants with genome editing compared to wild type (Feng et al., 2018).

Glycine max (L.) Merr.

Glycine max (soybean) contains important vegetable oils, proteins and bioactive secondary metabolites. Remarkably, isoflavonoids content in G. max is almost 100 times higher as compared to other leguminous plants, which have significant functions in plant disease resistance and human health (Budryn et al., 2018; Kant et al., 2019; Yu et al., 2003). As a signal molecule, isoflavonoids are involved in activating nod genes (Subramanian et al., 2006). Soybean phenylpropanoid pathway produces isoflavonoids by a sequential but complex process (Zhang et al., 2016). Briefly, the metabolic pathway for isoflavonoid biosynthesis starts by hydroxylation of flavanone which is catalysed by isoflavone synthase (IFS) which shares common substrate with flavanone-3-hydroxylase (F3H) and flavone synthase II (FNS II). GmIFS1 and GmIFS2 have been isolated from G. max (Jung et al., 2000). Zhang et al. (2020) studied multiplex CRISPR/Cas9 genome editing that was used to target GmF3H1, GmF3H2 and GmFNSII-1 in the hairy roots of G. max. Metabolomic analysis of GmF3H1, GmF3H2 and GmFNSII-1 triple mutants showed significant enhancement in isoflavone content. Additionally, the viral titre of Soybean mosaic virus (SMV) was significantly decreased. According to these findings, improved isoflavone content significantly enhanced soybean’s resistance to SMV (Zhang et al., 2020).

Humulus lupulus L.

Humulus lupulus, also called hops, contains phenols, bitter acids, prenylated flavonoids etc., which are considered to be medically significant for those suffering from diseases such as prostate and breast cancer, osteoporosis, menopause and anxiety (Mishra et al., 2020; Srečec et al., 2012). CRISPR/Cas9 was used for the first time to manipulate the gene expression of H. lupulus. Agrobacterium-mediated transformation [using the binary pKSE401] successfully edited 33.3% of the transformed plants. Targeted editing of a PDS gene in carotenoid biosynthesis pathway resulted in endogenous edited genes in H. lupulus and decreased concentrations of chlorophyll a/b and carotenoid pigments. The CRISPR/Cas9 system was cited as a precise way to target the genome sequence of hops (Awasthi et al., 2021).

Nicotiana tabacum L.

Nicotiana tabacum, a perennial herbaceous plant that produces tobacco, is reported to be a storehouse of important secondary metabolites such as alkaloids, terpenoids, flavonoids, phenylpropanoids etc. A number of recombinant products have also been produced using N. tabacum, including glycoenzymes α-galactosidase, glycohormone erythropoietin, proteases and xylanase (Jutras et al., 2020; Pantaleoni et al., 2014). Plants have been used for the production of pharmacological glycoproteins. These glycoproteins carry N-glycans with β(1,2)-xylose and a core α(1,3)-fucose, which have a significant influence on immunogenicity and allergenicity. CRISPR/Cas9 has been successfully used to knockout six glycosyltransferase genes in Nicotiana sp. It was used to produce recombinant proteins without core sugar α-1,3-fucose and β-1,2-xylose (Jansing et al., 2019). Gao et al. (2015) have projected that CRISPR/Cas9 system is a useful tool for targeted mutagenesis of N. tabacum genome mainly due to the high efficiency of this editing system in this species (Gao et al., 2015). Mercx et al. (2017) conducted the study on the knockout lines of XylT (β(1,2)-xylosyltransferase) and FucT (α(1,3)-fucosyltransferase) in suspended N. tabacum BY-2 cells. Expression cassettes for Cas9 and gRNA were driven by the 35S-PPDK and U6 transcriptional promoters, respectively, which knockout four FucT and two XylT genes. The IgG glycosylation profile was screened by mass spectrometry that displayed the presence of GnGn (69%), Man7 (9.3%) and GnM (4.8%) structure and lack of β(1,2)-xylose or α(1,3)-fucose on the glycosylation moiety (Mercx et al., 2017).

Papaver somniferum L

The benzylisoquinoline alkaloids produced by Papaver somniferum have clinical significance in biomedicine (Labanca et al., 2018). A genome editing strategy based on CRISPR/Cas9 genome editing has been applied to this plant successfully. CRISPR-SpCas9 type II was used to knockout 4′OMT2 (gene controlling benzylisoquinoline alkaloids biosynthesis). A resulting DSB was repaired by NHEJ, causing short indels to be introduced, resulting in gene dysfunction. Agrobacterium-mediated transformation was carried out utilising TRV (Tobacco rattle virus)-based synthetic binary plasmids expressing sgRNA and hCas9 (human-codon optimised Cas9)-encoding synthetic vector to inactivate 4′OMT2, thus regulating the biosynthesis of the BIAs. An HPLC-ToF/MS (high-performance liquid chromatography-time-of-flight mass spectrometry) study showed that P. somniferum plants with gene knockouts had significantly lower production of benzylisoquinoline alkaloids (Alagoz et al., 2016).

Salvia miltiorrhiza Bunge

Salvia miltiorrhiza, a traditional medicinal herb from China, contains diterpene compounds such as tanshinones and rosmarinic acid (RA) (Li et al., 2017a, 2017b; Shi et al., 2019). S. miltiorrhiza is extensively used against cardiovascular and cerebrovascular diseases and diabetes. Moreover, this plant has antioxidant and anti-inflammatory properties as well as cardioprotective and anticancer properties. According to earlier reports, an inactivation with the CRISPR/Cas9 system was shown to effectively target the SmCPS1 (diterpene synthase) gene from the tanshinone biosynthesis pathway. Rosmarinic acid synthase (RAS), an enzyme which catalyses RA biosynthesis, also accumulates lithospermic acid B (LAB) (Sander & Petersen, 2011). Agrobacterium rhizogenes-mediated transformation of hairy roots using CRISPR-Cas9 inactivated the SmCPS1 gene implicated in the production of tanshinone. An analysis of metabolite profiles in homozygous mutants revealed that tanshinone synthesis is completely absent, but these mutants produce the other phenolics. However, the tanshinone content of the chimeric mutants was reduced (Li et al., 2017a, 2017b). S. miltiorrhiza suspension cells were successfully transfected with Agrobacterium rhizogenes in order to knockout the SmRAS gene using CRISPR/Cas9 genome editing. One homozygous mutant, two heterozygous mutants and five biallelic mutants were generated using the Arabidopsis U6 promoter. Mutation in the SmRAS genes resulted in a decline in RA and LAB and an increase in 3,4-dihydroxyphenyllactic acid, a precursor of RA (Zhou et al., 2018). SmPAL1 was targeted and edited in the phenylpropane metabolic pathway using a software designed for CRISPR/Cas9 in the S. miltiorrhiza. SmPAL1-g1, SmPAL1-g2 and SmPAL1-g3 are three sequences which are likely CRISPR targets. These sequences achieved 53.3%, 76.6% and 10.0% enzyme digestion efficiencies, respectively (Qiu et al., 2018).

Challenges Associated with the Use of CRISPR/Cas Tools for Plant Natural Product Research

Off-Target Effects

Despite CRISPR/Cas9 being a popular tool among other genome editing tools due to its affordability, precision and simplicity, some negative consequences are also associated with it. The off-target effect is one such type. There are unintentional translocations, insertions, deletions, inversions and point mutations associated with off-target effects. Utilising the CRISPR/Cas9 tool, off-target mutations limit therapeutic implications. In lower group of organisms such as bacteria, a smaller number of off-target effects are reported because of a lower frequency of a spacer-PAM combination in their genome. There are a number of effective solutions to fighting off-target effects, such as mutations in the nuclease domain of Cas9 (Cho et al., 2014), dimerisation of nucleases (Fok1-dCas9) (Tsai et al., 2014) and CRISPR interference (Gilbert et al., 2014). There is considerable effort underway in higher eukaryotes to minimise off-target effects. Improved sgRNAs need to reduce mismatches (Doench et al., 2016) and restrict Cas9 levels in the cells to lessen off-target effects of these tools (Shen et al., 2019).

Editing Accuracy

The dominant error-prone NHEJ pathway for DSB repair is randomly generated small indels at the DSB locus. Unpredictable precision size of the indel leads to frameshift mutations. HDR creates more precise gene editing products. The efficiency of HDR is also reduced multiple folds due to the dominance of NHEJ pathway, regardless of whether an editing template is accommodated for HDR (Yang et al., 2020). In mammalian cells the efficiency of HDR is much lower (25%) than NHEJ (75%). To conquer this limitation, a few strategies have been effectively utilised to suppress the NHEJ pathway, for example, utilising the small molecule Scr7 on promoting HDR efficiency, as Scr7 binds to the DNA binding domain of ligase IV, KU70 or KU80 and thus inhibits with the of NHEJ pathway events (Li et al., 2017a, 2017b).

Efficient Delivery

The large size and positive charge of Cas9 protein and negative charge of sgRNAs make difficult the delivery of this protein into cell. The efficacious CRISPR/Cas9 application needs strong Cas9 and sgRNA delivery either by plasmid or by mRNA or by ribonucleoprotein (RNP) complex (Rahimi et al., 2020). Inappropriately, few medicinal plants discussed in this chapter lack efficient transformation methods. Nanoparticle-based delivery (polymeric nanocarriers, gold-based nanomaterials, metal-organic frameworks) is fetching a more striking approach due to its specific targeting and minimal exposure to nucleases (Carboni et al., 2019; Chen et al., 2020).

Genome Instability

DSBs may cause chromosomal translocations, instability of the genome which is a dangerous cellular event. CRISPR/Cas9 tool initiates DSB that may put the cells under severe stress. Cas9 expression in Streptomycetes leads to deletions in linear chromosomes (Hoff et al., 2018). CRISPR-based editing system (CRISPR-BEST), CRISPR interference (CRISPRi) and deaminase-based DNA base editors, that are not based on DSBs, have been conveyed as an effective genome editing tool (Eid et al., 2018; Tong et al., 2020).

Toxicity of Cas9

CRISPR-Cas9 tool is for high-throughput approach, but can be astonished by nuclease-induced toxicity because of DNA damage. Toxicity due to Cas9 expression has been reported in bacteria and green algae but has not yet been reported in plants. The presence of high intercellular Cas9 leads to toxicity. Reducing Cas9 levels using weak promoters or by controlling the size and frequency of target gene edits lead to a decreased toxicity (Dow et al., 2015; Morgens et al., 2017).

Indigenous Vs Introduced CRISPR’s Role

Bacteria and Streptomyces have evolved CRISPR/Cas systems which provide protection against foreign nucleic acids. A future research area of interest could be the interaction between native and introduced CRISPR/Cas.

Perspectives of CRISPR/Cas Genetic Engineering on Plant Natural Products

CRISPR-based genome editing has become a major breakthrough in the twenty-first century due to its effectiveness in knocking out. The success of this application prompted biochemical research companies to completely shift their focus to using CRISPR tools in plants for the production of sustainable agricultural crops and valuable plant natural products (Brinegar et al., 2017). It is crucial to efficaciously edit the genomes of medicinal plants. It enables accurate control of secondary metabolite pathways through regulating metabolic routes and removing rate-limiting constraints and feedback inhibitions. In order to meet the ever-expanding demand of biomedical industries, CRISPR/Cas genome editing may prove to be an exciting tool in metabolite engineering. For plants with a long life cycle, direct CRISPR/Cas-mediated editing can be ineffective. It may be extremely useful to employ microbial cell factories to substitute heterologous expression of secondary metabolic pathways. Earlier, CRISPR/Cas tool was mostly used to modify just one trait in somatic cells of higher plants through NHEJ. However, in recent years, CRISPR/Cas research mainly focused on the utilisation of homologous recombination for chromosomal rearrangement driven genetic traits in plants (Schindele et al., 2020). Incorporating artificial DNA sequences into plant genomes altering plant behaviour due to novel functions by CRISPR/Cas system is a promising approach to improve plant design and synthetic biology (Chen et al., 2019). Genome editing using CRISPR/Cas has immense potential to create biochemical factories, clone long DNA segments and manipulate biosynthetic pathways (Bennett-Baker & Mueller, 2017; Liu et al., 2015). Targeting long genomes with Cas9-aided targeting of CHROMOSOME (CATCH) and isolating those megabase-sized genomic fragments with CRISPR-mediated isolation (CISMR) are powerful tools for synthetic and chemical biology (Bennett-Baker & Mueller, 2017; Liu et al., 2015). Recent studies have shown that inducing artificial polyploidy via the CRISPR/Cas9 mechanism on A. rhizogenes hairy root cultures is a novel and promising way to increase secondary metabolite production in a wide range of medicinal plants (Niazian, 2019). As Agrobacterium-mediated transformation is expensive and time-consuming, tissue culture-free genome editing offers an alternative, which will likely be more efficient. The specificity of the Cas9-linked base editors has also been improvised through the expansion of the guide sequences sgRNAs and RNP-mediated delivery of the base editors. A genomic editing process has also been improved by enhancing the frequency of HDR to improve the efficiency and precision of the process. Despite receiving a lot of attention during the past several years, CRISPR/Cas has only just begun to make its way into natural product discovery. Few applications have been reported, and most of the work has focused on proving the feasibility of CRISPR/Cas systems in plants. The current CRISPR/Cas technology should be optimised, developed and innovated further, as no technology is perfect.

Conclusion

A number of plant secondary metabolites could potentially be used to treat several life-threatening human diseases. They provide protection against a wide range of diseases with a wide array of defence properties without causing harmful side effects. They are also relatively affordable and less costly than other methods of treatment. CRISPR/Cas9 has appeared as a breakthrough tool for metabolic engineering in plants. CRISPR/Cas9 modifies desired genomic parts by knocking out, knocking in, mutations etc. It is still in the early stages of CRISPR/Cas9-based genome editing in most medicinal plants as information about whole genomes and mRNAs sequences is lacking. Due to insufficient sequence information, this tool cannot edit key genes involved in plant secondary metabolite pathways. The limitations of this tool and strategic approaches to overcome them have already been discussed. However, a large number of secondary metabolites producing genes have not been modified yet with CRISPR/Cas9 for enhancing metabolite production. The use of higher plants as renewable sources of bioactive metabolites will be extended and enhanced through CRISPR/Cas9. In the future, we expect to achieve controllable and successful biotechnological production of valuable and as yet unknown plant phytochemicals through the continuation of efforts in this area.