Cyanobacteria are appealing photosynthetic hosts for chemical production that can be genetically manipulated to direct the native metabolic flux toward target chemicals of interest.<\/p>\n
CRISPR\/Cas9 and CRISPR\/Cas12a enable metabolic engineers to modify the genomes of cyanobacteria with gene substitutions, markerless point mutations, and gene knockouts and knock-ins with improved efficiency.<\/p>\n
The repression of native genes via CRISPRi affords a practical way of reducing the flux through competing metabolic pathways and directing it toward the target chemical.<\/p>\n
Syntheses of a range of industrial target chemicals have been demonstrated in cyanobacteria. To further diversify target chemicals and meet the demands of industry, attention needs also to be given to target chemicals that depend on metabolic pathways with poor flux or involve more complex metabolic routes.
\nIn metabolic engineering, the production of industrially relevant chemicals, via rational engineering of microorganisms, is an intensive area of research. One particular group of microorganisms that is fast becoming recognized for their commercial potential is cyanobacteria. Through the process of photosynthesis, cyanobacteria can use CO2 as a building block to synthesize carbon-based chemicals. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-dependent approaches have rapidly gained popularity for engineering cyanobacteria. Such approaches permit markerless genome editing, simultaneous manipulation of multiple genes, and transcriptional regulation of genes. The drastically shortened timescale for mutant selection and segregation is especially advantageous for cyanobacterial work. In this review, we highlight studies that have implemented CRISPR-based tools for the metabolic engineering of cyanobacteria.
\nMetabolic Engineering of Microbes
\nMetabolic engineering was first described more than three decades ago [1] by the late pioneer James E. Bailey as \u2018the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology\u2019 [2]. Typically, metabolic engineering focuses on chemical manufacturing applications though it can be extended to other applications such as bioremediation and biosensing [3, 4, 5]. Current industrial methods for the production or isolation of a vast majority of chemicals rely on the availability of fossil fuels or agricultural resources, which are unsustainable and leave a large ecological footprint (see Glossary) [6, 7]. By contrast, the field of metabolic engineering offers a sustainable and benign approach for chemical production [8].<\/p>\n
Photosynthetic organisms are ideal hosts for the production of chemicals because carbon can easily be obtained from CO2 present within the atmosphere, and energy from light can be harnessed to direct the carbon toward the syntheses of the target chemicals [9, 10, 11, 12]. Cyanobacteria are highly favored photosynthetic hosts due to their excellent amenability to genetic manipulations (Box 1). Over the past decade, a variety of chemicals have been synthesized in cyanobacteria using classical genetic approaches [13, 14, 15, 16, 17, 18, 19, 20]. However, only recently with the arrival of clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques new breakthroughs in the metabolic engineering of cyanobacteria have been made.<\/p>\n
Box 1<\/p>\n
+
\nBox 1
\nWhat Are Cyanobacteria and Why Are They Significant for Metabolic Engineering?<\/p>\n
CRISPR\/Cas9 Improves the Efficiency of Genome Editing in Cyanobacteria
\nIn cyanobacteria, genomic modification is a time-consuming affair and further made complicated by the fact that certain cyanobacteria are oligoploid or polyploid. For example, Synechocystis sp. PCC 6803 can harbor up to 53 chromosome copies per cell [21], whereas Synechococcus elongatus PCC 7942 can harbor as few as two copies per cell [22]. To create a homozygous mutant in an oligoploid or polyploid strain, a segregation procedure is therefore necessary to ensure all chromosome copies in the transformants carry identical sequences of the modified DNA. This involves multiple rounds of culture streaking aided by antibiotic selection, which can take several weeks to complete.<\/p>\n
The advent of CRISPR-based technologies has revolutionized the way genomes are edited. Box 2 summarizes the individual components of three major CRISPR systems. For cyanobacterial engineering, CRISPR\/CRISPR associated (Cas) systems are used alongside homologous recombination-dependent techniques. By inducing double-stranded cleavage of the genome, via CRISPR\/Cas, homologous recombination is stimulated in a DNA repair process known as homology-directed repair, resulting in an improvement in the efficiency of genome editing. This improvement in efficiency was first demonstrated in cyanobacteria by Wendt and colleagues [23] using the CRISPR\/Cas9 system. The nonbleaching protein A (nblA) gene was selected as the target because its mutation prevents depigmentation that would otherwise develop under nitrogen deprivation due to phycobilisome degradation. This is an easily observable phenotype that appears once all nblA copies are deleted. Thus, the nblA mutant serves as an excellent visual reporter for the process of segregation. In this study, Cas9 treatment led to an improved frequency of editing with approximately 70% of transformed cells arising from Cas9-mediated cleavage without the requirement for selection markers [23]. Moreover, CRISPR-based technologies can facilitate the segregation process. With only three rounds of streaking, full segregation was achieved within only 1 week. This improvement in efficiency was corroborated in a separate study, in which CRISPR\/Cas9 increased recombination efficiency in S. elongatus PCC 7942 by 57% [24]. Therefore, augmenting homologous recombination with CRISPR\/Cas systems greatly improves the genome-editing efficiency in cyanobacteria.<\/p>\n
Box 2<\/p>\n
+
\nBox 2
\nWhat Is CRISPR?<\/p>\n
CRISPR\/Cas9 Can Be Applied for Metabolic Engineering in Cyanobacteria
\nBy validating its usefulness in cyanobacteria, Li and colleagues [24] applied CRISPR\/Cas9 for the biological production of succinate, a bulk chemical with numerous industrial applications [25]. Under nitrate deprivation, much of the carbon flux in cyanobacteria is directed toward glycogen formation (Figure 1A,B). To divert the carbon away from glycogen and toward succinate, the glgC gene encoding glucose-1-phosphate adenylyltransferase essential for glycogen synthesis was deleted using CRISPR\/Cas9. In comparison with the wild-type, the glgC deletion mutant produced higher levels of succinate (0.18\u2005mg\/L). A knock-in of ppc and gltA genes within the glgC locus, again aided by CRISPR\/Cas9, to improve the tricarboxylic acid cycle supply of succinic acid precursors further increased succinate titers to 0.44\u2005mg\/L. These findings unequivocally demonstrated the feasibility of a CRISPR-based approach for the metabolic engineering of cyanobacteria.
\nAside from the increase in genome-editing efficiency, there are other practical advantages of using CRISPR\/Cas9 for genome editing in cyanobacteria. First, homology arms as short as 400\u2005bp are sufficient for homologous recombination and as effective as 700\u2005bp homology arms [24]. Usually, homology arms as long as 1000\u2005bp are needed to ensure adequate recombination efficiencies. In addition to shortening PCR protocols, shorter homology arms open up the possibility of integrating genes within smaller genomic target sites, thereby avoiding the risk of unwanted recombination events that could significantly alter the host behavior and phenotype. Second, CRISPR\/Cas9 significantly reduces the amount of template plasmid for homologous recombination from 2000\u2005ng to as little as 250\u2005ng [24].
\nCRISPR\/Cas12a Is a Compatible CRISPR System for Cyanobacterial Engineering
\nIn the studies by Wendt and colleagues [23] and Li and colleagues [24], high expression levels of Cas9 were observed to be toxic, resulting in reduced viability of S. elongatus UTEX 2973 and PCC 7942 cells, a phenomenon that has frequently been observed in eukaryotic organisms [26, 27]. This prompted Ungerer and Pakrasi [28] to explore the CRISPR Type V-A Cas12a (previously known as Cpf1) nuclease for genome editing in cyanobacteria (see Figure IB in Box 2). Knockout and single-point mutations were attempted in S. elongatus UTEX 2973 using nblA and psbA as the target genes, respectively, since these genes have well-known, observable phenotypes. For the knock-in approach, enhanced yellow fluorescent protein (eYFP) was used as the reporter for verifying transcriptional activity. In all cases, about 20% of the cells were correctly edited. Similar results were achieved for two other species, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120.<\/p>\n
For metabolic engineering work, native gene(s) need\/s to be targeted with a high degree of accuracy so as not to perturb adjacent genes or regulatory elements that may affect the production of the target chemical. In CRISPR-based editing, the target sequence depends on the position of the protospacer adjacent motif (PAM). Thus, Cas nucleases with different PAM specificities would allow greater flexibility in editing the cyanobacterial genome. In the case of Cas9 and Cas12a homologs, the canonical PAM sequences of 5\u2032-NGG-3\u2032 and 5\u2032-YTN-3\u2032 would permit targeting of GC-rich and AT-rich regions of the genome, respectively [29, 30, 31]. This is relevant because the average GC content of cyanobacterial genomes varies from 30.8% in Prochlorococcus MED4 to 68.4% in Vulcanococcus limneticus but with substantial exceptions; for example, in the latter, the GC content is 60.5% in the nif gene cluster encoding the apparatus for fixing atmospheric dinitrogen gas to yield ammonium [32]. Engineering biological nitrogen fixation into a non-diazotrophic photosynthetic organism provides a promising solution to the increasing demand for fixed nitrogen [33]. However, the number of required modifications is enormous. In addition to the 22\u201325-core nif genes, tight regulation of the expression of these genes is required, and the supply of cofactors, energy, and redox equivalents has to be secured where and when they are needed. CRISPR-based approaches provide a promising way forward to meet these challenges, which were considered intractable in the past.
\nGene Expression in Cyanobacteria Can Be Downregulated with dCas9
\nThe knockout or knock-in of genes can at times compromise the viability of the host organism [34]. In the case of cyanobacteria, the oligo-to-polyploidy states of some cyanobacterial species can make it arduous to obtain homozygous knockout mutants [21]. To compound the issue further, the cytotoxicity associated with high Cas9 induction necessitates preliminary experiments to fine-tune the expression of Cas9 [23]. Rather than utilizing CRISPR-based genome editing, CRISPR interference (CRISPRi) offers an alternative, viable approach for cyanobacterial engineering which relies on an enzymatically inactive dead Cas9 (dCas9). This is especially relevant for essential genes that cannot be deleted but only reduced in their expression. Yao and colleagues [35] first demonstrated CRISPRi in Synechocystis sp. PCC 6803, using GFP as a reporter. A small set of single-guide RNAs (sgRNAs) was inserted simultaneously in the slr2030-slr2031 neutral site, each one under the control of its own constitutive promoter [35]. As much as 94% repression of GFP was achieved with the anhydrotetracycline (aTc)-inducible promoter, PL22. This was found to be reversible upon removal of the inducer aTc [35]. Highest repression was observed when the sgRNA was targeted to the nontemplate strand close to the \u221235 promoter region and transcription start site [35]. By contrast, Behler and colleagues [36] showed that the essential RNase E gene in Synechocystis sp. PCC 6803 could be more effectively downregulated when the sgRNA targeting position was +7 to +27 within the coding sequence of the nontemplate strand. In a separate study, Huang and coworkers [37] maintained cells in a repressed state for 21\u2005days using CRISPRi. These studies clearly showcase the applicability of dCas9 for CRISPRi-based repression in cyanobacteria [35, 36, 37].<\/p>\n
One notable observation from the work by Yao and colleagues [35] was that repression of the target gene, irrespective of the strength of the promoter, occurred even in the absence of the inducer. This type of response may be problematic for the regulation of those genes that are essential for cell growth and maintenance. Gordon and colleagues [38] devised a CRISPRi system with an improved dynamic response that would allow tighter and greater control of repression. Using Synechococcus sp. PCC 7002 and the fluorescent YFP reporter, various promoters for sgRNA and dCas9 expression were assessed. The best dynamic range was observed when both dCas9 and sgRNA were expressed under the control of aTc-inducible promoters. CRISPRi-controlled transcriptional activity of YFP was found to be both reversible and titratable. Attempts to improve the dynamic range with alternative promoters or modifications of the sgRNA were unsuccessful. However, by reducing the strength of the ribosome-binding site, background levels of repression could be lowered to 30%, resulting in an improved dynamic response [38]. Within the context of cyanobacterial engineering, tighter induction systems will be critical for allowing greater dynamic control of metabolic pathways and improving the efficiencies of the production of the target chemical.
\ndCas9 Offers an Alternative Approach for Metabolic Engineering in Cyanobacteria
\nCRISPRi, rather than Cas9-dependent genome editing, offers a more pragmatic approach for metabolic engineering in cyanobacteria. Native genes can be repressed to reduce the carbon flux through competing metabolic pathways. This in turn results in a greater flux of carbon through the desired metabolic route and increases production of the target chemical. Yao and colleagues [35] first introduced a workable CRISPRi system in cyanobacteria by downregulating the production of polyhydroxybutyrate and glycogen. Metabolites such as polyhydroxybutyrate and glycogen serve as natural sinks for carbon storage. Therefore, reducing the flux through these pathways is an effective approach for improving the production levels of carbon-based target molecules in cyanobacteria. Repression of the phaE gene encoding a subunit of polyhydroxyalkanoate synthase effectively eliminated synthesis of polyhydroxybutyrate in Synechocystis sp. PCC 6803, while repression of glgC reduced glycogen synthesis by 80% [35]. Similarly, Huang and coworkers [37] reduced the glycogen content to 4.8% of the wild-type using S. elongatus PCC 7942 as the host. Furthermore, by downregulating glycogen synthesis, via repression of glgC, with the additional repression of sdhA and sdhB genes (Figure 1A,C), which encode for enzymes involved in the conversion of succinate to fumarate, succinate levels were enhanced 12.5-fold to 0.63\u2005mg\/L [37].<\/p>\n
CRISPRi-based engineering of cyanobacteria was also demonstrated by Gordon and colleagues [38], who experimented with native genes related to light harvesting in Synechococcus sp. PCC 7002. Repression of cpcB, encoding for a subunit of the phycobiliprotein complex, resulted in a severe reduction in the number of phycobilisomes [38]. Reducing the cyanobacterial light harvesting antenna size in this way is a promising strategy for increasing light penetration in dense cultures and improving photosynthetic efficiency [43]. The authors also demonstrated that repression of glutamine synthetase I (glnA gene), an enzyme that catalyzes the pivotal step between carbon and nitrogen metabolism, could be used to reduce nitrogen assimilation and increase the carbon flux toward lactate, a commonly used chemical agent within the food industry [38].<\/p>\n
Extending the range of strains and target chemicals, Higo and colleagues [44] utilized a CRISPRi approach for the engineering of Anabaena sp. PCC 7120. This strain fixes dinitrogen gas to yield ammonium. Nitrogen fixation proceeds in a specialized cell type known as heterocyst that differentiates from a vegetative cell [45, 46, 47]. Since only vegetative cells can be mutated directly, and not heterocysts, the use of heterocysts as production hosts presents a greater technical challenge than that of vegetative cells. Higo and colleagues [44] were able to show that ammonium production could be turned on and off in an inducer (aTc)-dependent manner for both vegetative cells and heterocysts by controlling the expression of glnA expression, via induction of dCas9 and sgRNA. Thus, CRISPRi-based engineering of cyanobacteria can be applied for the production of nitrogenous, as well as carbonaceous chemicals.
\nCyanobacterial Engineering Is Facilitated by Markerless Selection and Multiplexing
\nTo rationally optimize the production of a target chemical, a firm understanding of the metabolic flux of the native cyanobacterial pathways is often necessary. Kanno and coworkers [48] individually evaluated up to six native cyanobacterial genes to enhance the carbon flux toward the production of 2,3-butanediol, a bioplastics precursor. They achieved this by iterative modification of the genome, a process that depends on the use of markerless techniques. Conventional approaches for generating markerless mutants of cyanobacteria are laborious and time-consuming as the marker genes required for cell selection or counter-selection need to first be inserted and then removed from the host organism [49]. CRISPR-based editing, by contrast, does not require any selection or counter-selection genes based on cell viability. Instead, the selection process is based on cell viability. The cutting specificity of CRISPR permits cells with the correctly modified genome to remain viable, whereas cells with nonmodified or partly modified genomes become nonviable due to the lethality of double-stranded breaks [50]. This simplified selection process means that markerless cyanobacterial mutants can be obtained in as little as a week [23].<\/p>\n
Another useful feature of CRISPR is multiplexing, in which multiple genes can be targeted in a parallel, rather than in an individual manner [51]. As a proof-of-concept, CRISPR-based multiplexing was first shown for cyanobacteria by Yao and coworkers [35]. In this work, the research team knocked down four putative aldehyde reductases and dehydrogenases. The DNA sequences encoding for the various sgRNA molecules were inserted within the slr2030-slr2031 neutral site and expressed constitutively. Based on RT-qPCR analysis, as much as 50\u201395% repression was achieved. In a more recent study by Kaczmarzyk and colleagues [39], multiplexing was applied to good effect as part of a CRISPRi approach to redirect the fatty acid flux from membrane biosynthesis to fatty alcohol production in Synechocystis sp. PCC 6803. Up to six native genes were simultaneously repressed, each involved in the consumption of fatty acyl-ACP. With repression of all six genes, optimal production levels of the C16 fatty alcohol (1.1\u2005mg\/g dry cell weight) and the C18 fatty alcohol (9.3\u2005mg octadecanol\/g dry cell weight) were achieved. Multiplexing opens up the possibility of applying combinatorial approaches for the optimization of cyanobacteria, which in turn would permit greater coverage of the metabolic landscape. In summary, both the markerless and multiplexing features of CRISPR will undoubtedly accelerate the metabolic engineering of cyanobacteria.
\nKey Challenges in Engineering Cyanobacteria
\nBy invoking double-stranded breaks, CRISPR-based editing is a well-proven strategy for editing the genomes of many microbes, including cyanobacteria [23, 26, 40, 41, 52, 53]. Although genome-cutting tools such as meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs) have existed since the 1990s, their limited target specificity and requirement for customized engineering have precluded their use as tools for editing bacterial genomes in general [42, 54, 55]. As a result, TALENs and ZFNs have been reserved mainly for eukaryotic systems [55].<\/p>\n
A CRISPR-based system, by contrast, can elicit double-stranded breaks with a reasonable level of specificity while allowing sufficient flexibility with respect to genome target sites due to the increasing number of applicable Cas enzymes. In addition, CRISPR-based approaches require little a priori knowledge, multiple genetic modifications can be completed in parallel, guide RNA molecules can be custom synthesized at very low cost, and several rounds of genomic modifications can be completed within a week by simply altering the sequence of the guide RNA. These conveniences have led to the swift development of markerless and multiplex features of CRISPR-based tools. For these reasons, a CRISPR-based approach is likely to become the preferred genome-editing tool for cyanobacteria.<\/p>\n
Off-target effects of the CRISPR technique is an area of particular concern and may well lead to cytotoxic effects or other unwanted phenotypes [56]. Such effects, if any, remain to be investigated in cyanobacteria. Though CRISPR\/Cas9 nickases [57] or engineered Cas nucleases [58] with improved DNA specificity could provide the means for minimizing off-target sites, controlled induction of Cas9, as reported in several studies, could also possibly reduce off-target effects [28, 40]. In light of cytotoxicity issues, the use of Cas9 needs to be exercised with great caution, and its induction tightly controlled. Cas12a (Cpf1), by contrast, does not appear to exhibit toxic effects in cyanobacteria and may serve as a better alternative to Cas9. In addition, Cas12a requires only a single customized CRISPR array, of which the RNA is processed into individual sgRNAs by the Cas enzyme itself, whereas Cas9 depends on multiple sgRNA molecules that need to be cloned separately and each relying on its own promoter. The simpler mechanism and smaller-sized sgRNA for the CRISPR\/Cas12a system will help to reduce costs for the engineering of cyanobacteria.<\/p>\n
For CRISPRi approaches, the level of repression will be influenced greatly by the complementarity between the sgRNA and the target DNA. The general rule for achieving effective repression is that the sgRNA should target a DNA sequence close to the transcriptional start site on the nontemplate strand rather than on the template strand [59, 60]. Interestingly, Yao and coworkers [35] observed that one of the sgRNAs used in their CRISPRi displayed weak repression and hypothesized that the sgRNA may target a sequence that is distal from the transcription start site. Therefore, in practice, several different sgRNAs would need to be tested to implement the most effective sgRNA for repression of the target gene.<\/p>\n
Another key challenge that is pertinent to the engineering of cyanobacteria is the choice of the target chemical. In general, target chemicals with the highest productivities in cyanobacteria are those with relatively simple chemical structures that emanate from high flux pathways. These target chemicals are usually derived from pyruvate or directly from the Calvin cycle, and include ethanol (0.5\u2005g\/L\/day) [61], glycerol (1.3\u2005g\/L\/day) [62], and 2,3-butanediol (1.1\u2005g\/L\/day) [48]. Though such targets provide excellent fundamental insights into the metabolic flux of cyanobacterial pathways, the diversification of target chemicals, that depend on pathways with poor flux or involve complex metabolic routes, will be particularly important for meeting the demands of industry.
\nConcluding Remarks and Future Perspectives
\nSeveral studies have demonstrated the successful application of CRISPR-based techniques for the engineering of cyanobacteria [23, 24, 28, 35, 36, 37, 38, 39, 44]. The technical aspects of these studies are summarized in Table 1. As is the case with other microbial hosts, CRISPR-based techniques for cyanobacteria are aided with plasmids [26, 40, 41, 52]. Table 2 compares the features of conventional and CRISPR-based approaches for cyanobacterial engineering. \u2018Conventional\u2019 approaches refer to techniques used for engineering a deletion, insertion, or point mutation in cyanobacteria that rely on double homologous recombination between a suicide vector and the host genome, and involve the replacement of the gene of interest with a selective marker. There are no reported studies using ZNFs or TALENs in cyanobacteria. So far, CRISPR technology has been applied to engineering Synechococcus sp. PCC 7002 [38], S. elongatus PCC 7942 [24, 37] and UTEX 2973 [23, 28], Synechocystis sp. PCC 6803 [28,35,36,39], and Anabaena sp. PCC 7120 [28, 44]. CRISPR-based engineering has several benefits compared with conventional strategies. First, incorporating selection markers is unnecessary because selective pressure, based on cell viability, is integral to the CRISPR approach. Hence, CRISPR-based editing allows the creation of markerless knockouts and knock-ins, opening up the possibility of introducing or deleting an unlimited number of genes in a multiplexed manner. Second, avoiding the use of selection markers nullifies time-consuming downstream protocols to remove the selection marker once the editing has been performed and, furthermore, reduces the length of time for segregation. Third, with CRISPRi, fine-tuning metabolic pathways and reducing cellular fluxes toward unwanted side products are made possible without grossly affecting cell viability.<\/p>\n
Gene replacement: nblA or nifH knockout and simultaneous eYFP knock-in
\nMarkerless point mutation, knockout, knock-in, direct gene replacement using Cas12a
\nS. elongatus UTEX 2973, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120
\nFrancisella novicida Cas12a (Cpf1)
\npSL2680 (Addgene Plasmid #85581) based on RSF1010
\nKanamycin for S. elongatus 2973 and Synechocystis sp. PCC 6803, neomycin for Anabaena sp. PCC 7120, curing of plasmid to obtain markerless editing without antibiotics
\nTrans, inducible Plac for cas12a (cpf1), constitutive PJ23119 for CRISPR array
\n[28
\ndHR independence for dCas9 has not been experimentally verified in cyanobacteria due to gene stability risks associated with the use of plasmids.
\nOver the coming years, CRISPR-based approaches will greatly benefit the metabolic engineering of cyanobacteria. The strategies adopted in cyanobacteria have included knockouts, knock-ins, and downregulated transcriptional activity of target genes. Other possible strategies that have yet to be attempted include the upregulation of transcriptional activity, via CRISPRi, and regulation of mRNA levels, possibly via the Cas13a system [63, 64]. So far, target genes have been limited to those that encode for enzymes involved in the biosynthesis of the desired chemical, or unwanted metabolites generated from competing pathways. Derepression of native pathways, via disruption of genes that serve regulatory roles, is another effective strategy that is yet to be employed in cyanobacteria. Free fatty acid production in Synechocystis sp. PCC 6803 could, for example, be enhanced with deletion of transcriptional regulators such as cyAbrB2 [65], or even post-transcriptional regulators such as IsaR1 [66] or PsrR1 [67]. Directing the native metabolic flux toward the desired product increases both the production level and efficiency of the target product.<\/p>\n
So far, very few cyanobacterial studies have demonstrated the multiplex feature of the CRISPR technology. The highest number of genes that have been edited in parallel so far is six [39]. Since the metabolic optimization of cyanobacteria to produce a target chemical is quite likely to involve numerous genetic and regulatory factors, multiplexing needs to be employed more widely in the future [35, 39].<\/p>\n
Increased understanding of various CRISPR mechanisms and systems will undoubtedly inspire more advanced approaches for the engineering of biological hosts such as cyanobacteria. Considering the benefits of CRISPR-based approaches for metabolic engineering, the characterization of CRISPR components from diverse organisms would be an excellent strategic direction for researchers to pursue over the upcoming years [68, 69, 70]. The potential of CRISPR-based technologies for the metabolic engineering of cyanobacteria cannot be overestimated (see Outstanding Questions). Providing researchers with a greater choice of CRISPR-based tools, each with subtly varying properties and mechanisms, will help to drive innovative techniques and strategies for the engineering of not only cyanobacteria, but also other biotechnologically relevant organisms.<\/p>\n","protected":false},"excerpt":{"rendered":"
Cyanobacteria are appealing photosynthetic hosts for chemical production that can be genetically manipulated to direct the native metabolic flux toward target chemicals of interest. CRISPR\/Cas9 and CRISPR\/Cas12a enable metabolic engineers to modify the genomes of cyanobacteria with gene substitutions, markerless point mutations, and gene knockouts and knock-ins with improved efficiency. The repression of native genes […]<\/p>\n","protected":false},"author":3,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_seopress_robots_primary_cat":"","nova_meta_subtitle":"","footnotes":""},"categories":[5572],"tags":[5796,12051,14647],"supplier":[11310],"class_list":["post-54005","post","type-post","status-publish","format-standard","hentry","category-bio-based","tag-biotechnology","tag-chemicals","tag-engineering","supplier-cell-magazine"],"_links":{"self":[{"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/posts\/54005","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/comments?post=54005"}],"version-history":[{"count":0,"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/posts\/54005\/revisions"}],"wp:attachment":[{"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/media?parent=54005"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/categories?post=54005"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/tags?post=54005"},{"taxonomy":"supplier","embeddable":true,"href":"https:\/\/renewable-carbon.eu\/news\/wp-json\/wp\/v2\/supplier?post=54005"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}