Anthropogenic emission of carbon dioxide (CO₂) has driven an unprecedented high level of CO₂ in the atmosphere, leading to an approximately 1.1°C increase in the average global temperature (Tollefson, 2021). This increase has reached an alarming level and has caused global and local climate issues. It also leaves a very small window to achieve the 1.5°C target settled in the Paris Agreement and reinforced in the UN Climate Conference in Glasgow (COP26). The atmospheric CO₂ level must be lowered by a significant amount by controlling the emission of CO₂ and sequestering CO₂ from the atmosphere at the same time. In this endeavor, biotechnology provides promising routes. In one way, carbon-neutral biotechnology utilizes sustainable carbon sources (e.g., agriculture and forest wastes) to produce chemicals (e.g., ethanol, butanol, and 2,3-butanediol), alleviating the reliance on fossil fuels and reducing CO₂ emissions (Liu et al., 2020). In another way, carbon-negative biotechnology directly consumes industrial or atmospheric CO₂ for the bioproduction of fuels and value-added chemicals (Liu et al., 2020; Liew et al., 2022).

With metabolic engineering and synthetic biology, the inventory of products from biological routes has been greatly expanded, and the production and yield have been improved. For instance, the baker’s yeast Saccharomyces cerevisiae has been genetically engineered to convert lignocellulosic feedstock to bioethanol and chemicals, exhibiting the potential of carbon-neutral biotechnology (Wei et al., 2013; Sun et al., 2021). Recently, S cerevisiae has been engineered to utilize wasteful CO₂ accumulated during lignocellulosic sugar fermentation by the installation of a CO₂ fixation pathway, transforming the correlated biotechnology from a carbon-neutral process to a carbon-negative technology (Li et al., 2017; Xia et al., 2017). Inspiringly, Gassler et al. (2020) generated an engineered yeast Pichia pastoris capably of growing with CO₂ and methanol, opening a new window for heterotrophic yeast to use one-carbon (C1) compounds as sole carbon sources. Similar enterprises have been made in Escherichia coli, and artificial autotrophic E. coli has been generated via the implementation of CO₂ fixation pathways and adaptive laboratory evolution (Antonovsky et al., 2016; Gleizer et al., 2019; Flamholz et al., 2020).

Another biological path is to employ microorganisms that metabolize CO₂ innately, such as photoautotrophic cyanobacteria and chemoautotrophs, including acetogens and methanogens. These organisms can use CO₂ as a carbon source from either industrial waste gases or the atmosphere (Fackler et al., 2021). CO₂-metabolizing microorganisms have shown great potential as microbial chassis, and industrial attempts have been made (Liu et al., 2020; Liew et al., 2022). Given the advances in synthetic biology, these microbes play more important roles on the path towards a sustainable future with enhanced CO₂ utilization efficiency and an expanded spectrum of products. For instance, cyanobacterium Synechocystis sp. PCC 6803 has been modularly engineered to produce a high titer of 1-butanol, short/medium-chain carbohydrate, and lactate from CO₂ (Liu X. et al., 2019; Shabestary et al., 2021; Yunus et al., 2022). Lately, a pioneer study conducted by LanzaTech, Inc. (Skokie, IL, United States) shows that Clostridium autoethanogenum can convert syngas (consisting of CO₂, CO, and H₂) to acetone and isopropanol, and a pilot-scale fermentation in a 125-L scalable reactor was demonstrated (Liew et al., 2022). These advances have validated the capability of CO₂-metabolizing chassis in the fixation of CO₂ and production of value-added chemicals, and these succuss illustrated the ever-increasing power of synthetic biology in biotechnology.

CRISPR-Cas systems, the bacterial and archaeal immune systems, have been repurposed as synthetic biology tools for gene editing and regulation (Knott and Doudna, 2018). They have been revolutionizing biotechnology in fundamental ways. Though still in its infant stage, multiple CRISPR-Cas-based synthetic biology tools have been developed for cyanobacteria, acetogens, and methanogens, driving the rising of novel biotechnologies based on CO₂-metabolizing microbes. Herein, we summarize the current progress of CRISPR-Cas systems in genetically engineering microbial CO₂-metabolizing chassis, especially cyanobacteria, acetogens, and methanogens, for the conversion of CO₂ to biofuels and value-added products, and we discuss the challenges and future endeavors in developing more efficient synthetic biology tools.

Microbial CO₂-metabolizing chassis, mainly autotrophic microorganisms, can use CO₂ as a sole carbon source for catabolic and anabolic activities. Until now, six CO₂ fixation pathways have been identified, among which the Calvin cycle and Wood-Ljungdahl Pathway are the most understood and applicable routes (Fuchs, 2011; Muller, 2019; Gleizer et al., 2020). Compared to the complementary physiochemical strategies, biological ways have advantages in forming carbon-carbon bonds with one-carbon (C1) building blocks using either solar energy or redox power from inorganic compounds, i.e., iron, sulfide, and ammonia, offering better opportunities for bioproduction by the microbes themselves or in combined biotic-abiotic processes (Li et al., 2012; Sakimoto et al., 2016; Jin et al., 2021).

Photoautotrophic organisms, such as microalgae and cyanobacteria, can fix CO₂ with solar energy through the Calvin cycle and produce a large variety of organic compounds. Notably, efforts have been made to design and build engineered cyanobacteria for the production of biofuels (i.e., biodiesel, bioethanol, and isobutanol), value-added chemicals (Santos-Merino et al., 2019; Xia et al., 2019), and food-related products, such as starch (Luan et al., 2019). Besides the photoautotrophs, Cupriavidus necator (formerly Ralstonia eutropha), a facultative chemolithotroph, can grow on CO₂ through the Calvin cycle as well with H₂ or formate as the electron donor without light. C. necator has also been engineered as a novel chassis for bioproduction (Li et al., 2012; Panich et al., 2021). Chemoautotrophic organisms harboring the Wood-Ljungdahl Pathway can utilize CO₂ and H₂ anaerobically. As the key representatives, acetogens, especially strains from the class of Clostridia (e.g., C. autoethanogenum, Clostridium ljungdahlii, Acetobacterium woodii, and Eubacterium limosum), have been interrogated and engineered to utilize CO₂ (with H₂) or CO₂-containing mixed gases (Muller, 2019; Fackler et al., 2021). Due to the requirement and capability of co-utilization of H₂, acetogens can also be the bridge connecting bioproduction and “Power-to-Gas” technology, generating a novel nexus “Power-to-X” (Molitor et al., 2019; Mishra et al., 2020). Similarly, methanogens play important roles in different bioprocesses in various niches, such as in the gut, in soil, and in engineered systems (i.e., wastewater treatment facilities). They produce CH₄ from CO₂ and H₂ or other one-carbon compounds, like methanol (Thauer et al., 2008; Zabranska and Pokorna, 2018). As methanogens are archaea, they typically possess unique industrial merits, including high tolerance to temperature and osmatic stress, making them advantageous CO₂-metabolizing chassis.

CRISPR-Cas-based synthetic biology tools are repurposed from the bacterial and archaeal immune systems (Jiang and Doudna, 2017), and the innovations in CRISPR-Cas-based systems have been reshaping biotechnology in fundamental ways (Knott and Doudna, 2018; Pickar-Oliver and Gersbach, 2019). For instance, an artificial autotrophic P. pastoris was generated via the integration of six foreign genes and deletion of three innate genes with the CRISPR-Cas-based gene-editing tool (Gassler et al., 2020). Recently, CRISPR-Cas-based methods have also been deployed to upgrade carbon-negative bioprocess by manipulating CO₂-metabolizing chassis. In this section, we focus on the adaption of different CRISPR-Cas systems for the perturbation of CO₂-metabolizing microbes. We highlight achievements and challenges in cyanobacteria, acetogens, and methanogens.

CRISPR-Cas-based genome editing in microbes typically has two steps: RNA-guided DNA cleavage and DNA repair of the double-strand break, the latter of which eventually resulted in the editing of a target gene (Selle and Barrangou, 2015; Knott and Doudna, 2018). Taking the Class II Type II CRISPR-Cas system from Streptococcus pyogenes as an example, the single CRISPR effector Cas9 is led by a guide RNA (gRNA) consisting of a targeting sequence (spacer), which is complementary to the target sequence (protospacer), a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracer RNA) (Jiang and Doudna, 2017). When the complex of Cas9 and gRNA reached the target sequence, it recognizes the protospacer when a protospacer adjacent motif (PAM) presents. Then, the Cas9 nuclease cleaves the DNA and leaves a double-strand break, generating a “dead or alive” scenario for the microbe (Vento et al., 2019). With DNA repair mechanisms, the target gene will be edited to survive the deadly cleavage of Cas9 (Figure 1A). CRISPR-Cas-based genome editing has been prosperous due to the ease of use and the clean editing products without leaving a marker or a scar.

In cyanobacteria, CRISPR-Cas-based genome editing has been realized (Xia et al., 2019). Li et al. (2016) adapted a CRISPR-Cas genome editing tool for cyanobacterium Synechococcus elongatus PCC 7942 based on S. pyogenes Cas9 with a transient expression system. Later, a plasmid-based CRISPR-Cas system was developed for S. elongatus UTEX 2973, a fast-growing cyanobacterium showing great potential in sustainable bioproduction (Wendt et al., 2016). However, these countable successes implied the severe toxicity of Cas9 on cyanobacteria. Due to the toxicity of Cas9, conventional CRISPR-Cas-based genome editing tools with a “dead or alive” selection have not been thriving in cyanobacteria. To surmount this bottleneck, two strategies have been engaged. One is to use alternative Cas proteins. For instance, the Class II Type V CRISPR system with Cas12a as the effector showed lower toxicity than Cas9 to cyanobacteria. By using Cas12a, Ungerer and Pakrasi (2016) achieved CRISPR-Cas-based gene editing in S. elongatus UTEX 2973, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7102. Another way is to control the expression of the CRISPR-Cas system tightly. Hudson and colleagues hired a tightly regulated RNA device, the theophylline-responsive riboswitch, to maintain a low enough OFF-state expression of Cas9 to prevent its toxicity, and induce the genome-editing when required (Cengic et al., 2022). By applying this system, the reliable transformation of a replicable plasmid harboring CRISPR-Cas9 was obtained, leading to successful deletions and insertions of DNA fragments in the genome of Synechocystis. To our best knowledge, this study also reported multiplex genome editing in cyanobacteria for the first time regardless of methods. As multiple genes are typically involved in engineering a microbe for desired functionalities, multiplexing is of great importance in synthetic biology by saving considerable time and labor.

CRISPR-Cas-based genome editing tools have also been established in acetogens and methanogens. For acetogens, Cas9 and Cas12a based methods have been devised in C. autoethanogenum (Nagaraju et al., 2016), C. ljungdahlii (Huang et al., 2016; Zhao et al., 2019), and Eubacterium limosum (Shin et al., 2019). To be noted, enhanced genome editing was achieved via a combination of CRISPR-Cas and serine recombinase (Huang et al., 2019). As reported, a phage serine recombinase was used for the integration of large DNA fragments while CRISPR-Cas inserts a small recognition motif of the recombinase. With this method, a butyric acid production pathway was successfully introduced to C. ljungdahlii for the production of butyric acid from syngas (Huang et al., 2019). Similar to the abovementioned method for cyanobacteria, a tightly regulated system controlled by a riboswitch, namely RiboCas, was designed to enable CRISPR-Cas-based genome editing in Clostridium strains, including Clostridium pasteurianum, Clostridium difficile, and Clostridium sporogenes (Canadas et al., 2019). Moreover, CRISPR-Cas-based deletion and integration were accomplished in methanogen Methanosarcina acetivorans via applying an inducible CRISPR-Cas9 system from S. pyogenes (Nayak and Metcalf, 2017). In the same study, the authors reported CRISPR-based deletion via the implementation of a foreign non-homologous end-joining (NHEJ) machinery from Methanocella paludicola, enabling the deletion of gene fragments (75–2.7 kb) without repairing DNAs. Recently, CRISPR-Cas-based genome editing was reported in methanogenic archaea Methanococcus maripaludis with the Cas12a system from Lachnospiraceae bacterium (Bao et al., 2022), further expanding the genome editing tools for methanogens.

Actually, the toxicity of Cas proteins is not an exclusive issue in cyanobacteria, while most bacteria suffer from the toxic effects of CRISPR-Cas systems (Banno et al., 2018; Canadas et al., 2019; Vento et al., 2019). Despite screening alternative CRISPR-Cas systems and implementation of fine control modules, one alternative way is to employ the nuclease deactivated Cas protein (dCas) (Figure 1B). When the nuclease activity of Cas proteins is dead (dCas) or partially dead (nCas), dCas or nCas proteins are less toxic to bacteria compared to fully functional Cas effectors. By using dCas9, the resulting CRISPR-Cas system will no longer cleave the target DNA sequence but bind to the target. When dCas9 binds to the promoter or the coding sequence of a gene of interest, it will prevent the binding of RNA polymerase (RNAP), thus silencing the target gene at the transcription level and generating the method CRISPRi (Figure 1C) (Larson et al., 2013; Qi et al., 2013).

Due to the alleviated toxicity, CRISPRi obtained more popularity and has been developed for cyanobacteria. For instance, Yao et al. (2016) employed dCas9 to enable multiplex CRISPRi in Synechocystis sp. PCC 6803, Choi and Woo (2020) demonstrated CRISPRi with dCas12a in S. elongatus PCC 7942, and a dCas12a-based CRISPRi system was also established for S. elongatus UTEX 2973 (Knoot et al., 2020). In the former two reports, multiplex CRISPRi was achieved, and up to four genes were repressed at a single time, showing the huge potential of CRISPRi in engineering cyanobacteria for sustainable production (Yao et al., 2016; Choi and Woo, 2020). Notably, a CRISPRi system was devised for generating a gene repression library in Synechocystis sp. PCC 6803 in order to interrogate the genotype-phenotype interactions. By doing so, an industrially relevant strain with higher production of lactate, as a proof of principle, was engineered via CRISPRi-based repression of correlated essential genes (i.e., gltA and pcnB) related to lactate synthesis (Yao et al., 2020). More recently, CRISPRi was programmed as a genetic switch between cell growth and product synthesis. Shabestary et al. (2021) employed CRISPRi-based gltA regulation in a lactate-producing Synechocystis and achieve a high yield of lactate by decoupling cell growth and lactate production.

CRISPRi displays potential for acetogens and methanogens as well (Dhamad and Lessner, 2020; Fackler et al., 2021). Woolston et al. (2018) developed an inducible dCas9-based CRISPRi system for the repression of essential genes related to carbon metabolism in Clostridium ljungdahlii. Specifically, the pta gene encoding the phosphotransacetylase and the aor2 gene encoding the aldehyde:ferredoxin oxidoreductase were repressed with CRISPRi individually and in a multiplex mode, redirecting carbon from acetate to the desired product 3-hydroxybutyrate with significantly increased titer and yield (Woolston et al., 2018). In a pioneer study, a CRISPRi system was developed for archaeal methanogen M. acetivorans by applying S. pyogenes dCas9, and the system was evaluated by interrogating the gene cluster related to nitrogen fixation (nif operon) and its regulator (nrpR1) (Dhamad and Lessner, 2020).

Besides gene repression, transcriptional activation can also be possible with CRISPR activation (CRISPRa) (Bikard et al., 2013; Liu Y. et al., 2019). CRISPRa deploys a combination of dCas protein and transcription factor, such as the ω subunit of the RNAP. When CRISPRa targets the upstream of a promoter, it will help bring RNAP and activate the transcription of the corresponding gene (Figure 1C). Bikard et al. (2013) first developed a CRISPRa system for bacteria and employed the dCas9-ω fusion to allow upregulation of gfp and lacZ expressions in E. coli. Recently, more advances in CRISPRa have been reported in bacteria (Liu Y. et al., 2019; Schilling et al., 2020; Kiattisewee et al., 2021; Villegas Kcam et al., 2021; Tickman et al., 2022). Given these advances, new transcriptional factors have been systematically screened, and the application has been expanded from E. coli to other bacteria, including Paenibacillus polymyxa (Schilling et al., 2020) and Pseudomonas putida (Kiattisewee et al., 2021). Moreover, a full range of gene regulation from repression to upregulation has been achieved by programmable CRISPRi/a circuits (Tickman et al., 2022) and by designing the targeting loci (Liu Y. et al., 2019). Though not been adapted yet, the application of CRISPRa and CRISPRi/a circuits will be a powerful tool for gene regulations in CO₂-metabolizing microbes.

In this review, we summarized recent advances in developing and applying CRISPR-Cas systems for CO₂-metabolizing chassis. CRISPR-Cas-based genome editing and CRISPRi, have been reported in these microbes, and the methods have been advancing biorefinery and bioproduction with CO₂ as the carbon source, exhibiting great potential in alleviating CO₂ emissions and in reducing atmospheric CO₂ levels. However, more efforts are imperative to awake the full power of CRISPR-Cas systems in these CO₂-metabolizing chassis. CRISPRa, base editing, prime editing, and transposon-mediated integration may offer encouraging future directions in developing novel CRISPR-Cas systems for CO₂-metabolizing microorganisms. Moreover, discoveries of new CRISPR-Cas systems with special properties (e.g., a thermostable Cas9) are needed to engineer CO₂-metabolizing microorganisms, such as thermophilic strains Thermoanaerobacter kivui and Methanothermobacter thermautotrophicus (Moon et al., 2019; Fink et al., 2021).

Besides CO₂, CO, methane, methanol and formate are also important greenhouse gases and C1 compounds that can be obtained from waste gases and products or byproducts of clean energy industries. As such, natural and engineered C1-metabolizing microbes, including but not constrained in the autotrophs discussed here, will also be favorable microbial chassis for sustainable bioproduction. The development of novel synthetic biological tools, such as CRISPR-Cas systems, for C1 metabolizing organisms, will significantly foster innovations in carbon-negative biotechnologies.