Revolutionizing biofuel generation: Unleashing the power of CRISPR-Cas mediated gene editing of extremophiles

Molecular biology techniques like gene editing have altered the specific genes in micro-organisms to increase their efficiency to produce biofuels. This review paper investigates the outcomes of Clustered regularly interspaced short palindromic repeats (CRISPR) for gene editing in extremophilic micro-organisms to produce biofuel. Commercial production of biofuel from lignocellulosic waste is limited due to various constraints. A potential strategy to enhance the capability of extremophiles to produce biofuel is gene-editing via CRISPR-Cas technology. The efficiency of intracellular enzymes like cellulase, hemicellulose in extremophilic bacteria, fungi and microalgae has been increased by alteration of genes associated with enzymatic activity and thermotolerance. extremophilic microbes like Thermococcus kodakarensis, Thermotoga maritima, Thermus thermophilus, Pyrococcus furiosus and Sulfolobus sp. are explored for biofuel production. The conversion of lignocellulosic biomass into biofuels involves pretreatment, hydrolysis and fermentation. The challenges like off-target effect associated with use of extremophiles for biofuel production is also addressed. The appropriate regulations are required to maximize effectiveness while minimizing off-target cleavage, as well as the total biosafety of this technique. The latest discovery of the CRISPR-Cas system should provide a new channel in the creation of microbial biorefineries through site- specific gene editing that might boost the generation of biofuels from extremophiles. Overall, this review study highlights the potential for genome editing methods to improve the potential of extremophiles to produce biofuel, opening the door to more effective and environmentally friendly biofuel production methods.


Introduction
The emergence of petroleum-based fuel had an amazing impact on industrial growth, infrastructural development, power generation and several other such concepts. Fossil fuels now provide over eighty percent of the world's energy. The high usage and demand of fossil fuels however is concomitant with an ever increasing environmental and economic global concern for its sustainable production and usage. In addition to this, fossil fuels have several detrimental impacts on earth, its complex ecosystems, climate change and on humans. They generate more than 75% of global greenhouse gases and approximately 80% of carbon dioxide emissions exacerbating global warming. Even when the problems and disadvantages are clear and well understood, numerous reports show that the production of oil, gas and coal will practically double by 2030. By substituting conventional fossil fuels with biofuels that may be made from renewable organic material, many of these disadvantages can be reduced. It is important to promote renewable energy sources like biofuels, since they offer a more economical and environmentally friendly alternative (Darda et al., 2019). This alternative will also be beneficial in future logistics, and warehouse industries, requiring minimal mechanical changes (Singhal et al., 2022). Many microbial strains are used for biofuel production by fermentation such as Saccharomyces cerevisiae. This fungus is widely used for ethanol production via fermentation of monosaccharides on a commercial level. Also, different bacteria such as Clostridium thermosaccharolyticum, Zymomonas mobilis, C. thermohydrosulfuricum, Thermoanaerobacter brockii, T. mathranii, and T. ethanolicus are employed for fermentation. Biofuel production greatly depends on the microbial strains. Fortunately, these can be improved by site-specific genome editing (Adegboye et al., 2021). Current technologies, such as biochemical and molecular engineering, bioengineering, and fermentation engineering, can play a vital role in improving biofuel production. Due to the simplicity and efficiency of fermentation, microorganisms are recognized as promising sources for biofuel synthesis via numerous metabolic processes, including glucose catabolism, lipids oxidation, and the isoprenoid pathway (Joshi et al., 2022). Despite this, the fermentation of microorganisms can generate certain by-products that can lead to the impairment of the desired products. This issue is quite prevalent in large-scale manufacturing systems, which in turn has called for a drastic change in their current strategy of efficient and cost-effective biofuel production. With the continuous advance of genetic and genome engineering, it is now possible to implement various microbial strains to utilize the mechanism of hydrolysis which can convert complicated substrates into simple fermentable forms (Celińska et al., 2021).
The widely used and studied CRISPR-Cas system can be used to mediate selective genetic manipulation in industrial microbial strains such as the aforementioned to achieve optimal biofuel production. CRISPR-Cas technology, has revolutionized the use of gene editing tools. Biorefineries is one of the many fields, CRISPR-Cas has influenced. This tool is both easy and precise and could enable modification of different components in biofuel production, such as better hydrolysis, enhanced suppressing, and the targeting of competing metabolic routes. Compatibility with solvents and substrate utilization are two factors that will have a significant impact on future commercial successful applications.
The purpose of this review study is to investigate whether or not the editing of genomes using CRISPR-Cas technology might result in an increase in the amount of biofuel that is produced. Particular attention is being paid to the use of CRISPR-Cas in thermophilic microorganisms and the ways in which genetic editing might improve the organisms' capacity for the production of biofuels. The purpose of this study is to examine the impacts that genome editing has on metabolic pathways, as well as the attempts that have been made to limit off-target effects, and the overall influence that this has on the production of biofuels. In addition, this article illustrates the eco-friendliness of biofuels and the ways in which CRISPR-Cas technologies might help make the production of large-scale industrial biofuels less harmful to the environment.
The originality of this review study resides in the fact that the author chose to concentrate specifically on the use of CRISPR-Cas technology in extremophiles for the generation of biofuel. It underlines the promise of tools for genetic engineering, in particular CRISPR-Cas, to enhance several elements of biofuel production, such as the efficiency of hydrolysis, the inhibition of metabolic pathway, and substrate utilisation. This review also discusses the difficulties that come with manipulating genes in a way that produces off-target effects and investigates potential solutions to these problems. The effects of off-target genetic manipulation in metabolic pathways and efforts of reducing these will also be discussed. In this research, insights into the potential of CRISPR-Cas technology for the production of sustainable biofuels are provided by highlighting the environmentally friendly nature of biofuels and the role that CRISPR-Cas plays in enhancing the total output of biofuels.

Data collection and analysis
One hundred thirty publications on biofuels, pretreatment techniques, CRISPR-Cas, gene editing, and off-target impact were found in this review's Web of Science™ Core Collection. The non-commercial visualisation of similarities (VOS) viewer VOS viewer 1.6.18 (https:// www.vosviewer.com/) created the graphical mapping of the term cooccurrence and co-authorship. Future research orientations may be strongly predicted by the amount of published material. Different academic disciplines may be categorized using a keyword list. Fig. 1 displays maps of the co-citation of the most-cited terms from 130 publications published between 2018 and 2022 using network visualisation, density visualisation, and overlay visualisation techniques. The bulk of recent study has been devoted to developing new biofuels, and it's been stated that one of the most extensively researched areas with potential uses is genetic manipulation by CRISPR-Cas.

1st generation
First generation biofuels are produced by corn, wheat, soybeans and other such feedstock. The sources can separated to starch and sugar crops and oil seeds respectively for the manufacture of bioethanol and biodiesel (Fig. 2). The carbohydrates obtained from these types of feedstock as well as in the sources of other generations, can undergo fermentation with specific enzymes produced by microbes that excel in pentose sugar metabolism to bioethanol. The production of biodiesel is also sought out after, through the trans-esterification of straight vegetable oils. Their cheap production allows for under-developed countries to have a near inexhaustible form of fuel (Alaswad et al., 2015). The use of first generation biofuel sources come with numerous disadvantages, however. First and foremost, there is a significant competition with agriculture for the available land needed for cultivation. This has caused a problem of food vs biofuel to arise. Furthermore, to produce the biomass for biofuel production intense use of fertilizers and agrochemicals are used. This has led to the reduction in water and soil quantity further aggravating the problem of food availability. There are several other drawbacks of first generation sources. Greenhouse gas emissions, deforestation, biodiversity loss etc. further increase the environmental and social negative impact of food crops being used as biofuel. To lessen these effects, a less expensive and readily available biomass was needed. The answer lay in the waste produced from municipalities, agriculture and forests (Ho et al., 2014).

2nd generation
Grass, wood, and other organic resources are utilised as the feedstock for making biofuels instead of food crops. These non-food and nondigestible lignocellulose sources differ in their chemical composition. Their abundance of polysaccharides, hemicellulose and lignin which increases the complexity of their metabolism due to a higher resistance of being broken down. Nonetheless, this source can alleviate the competition for agricultural land, further reduce the costs of raw materials and competition for food production. One of the biggest drawbacks is the pretreatment of these carbohydrates. It is a costly endeavor and requires enzymes with varying specificities. Other problems include spatial distribution, the uncertain availability of the biomass, storage and transport. Thus, this feedstock supply has not yet been proven efficient for large-scale commercial use and production. To mitigate some of these disadvantages, strategies for the acquisition of biomass went from land to sea (Ho et al., 2014).

3rd generation
The concept of using algae as a feedstock source became extremely favored since they can grow in various aquaeous environments such as coastal seawater, saline water, wastewater and on wastelands. A few of the many advantages of algae is their amazing biomass production eclipsing the harvesting potential of the aforementioned sources. Moreover, they possess insignificant amounts of lignin and hemicellulose resulting in easier, quicker and more efficient hydrolysis. Apart from the distinct bioethanol and biodiesel types of fuel, biohydrogen and biomethane can be used for several purposes such as fuel cells and heating respectively. Microalgae feedstock is still in its infancy however. There are many challenges to perfect their production which include their cultivation, reaction vessel design and downstream treatment action. To achieve the desired biomass requires a relatively high investment which is a non-trivial trade-off. The unfortunately, necessity of fossil fuels for their production is another negative aspect to take into consideration. Even though the use of algae as a biofuel source option is not entirely promising at present, creating a robust and novel course of action could prove to be very beneficial for biofuel production in the coming future.

4th generation
The last and most recent generation of biofuels builds upon its predecessor to enhance efficiency and increase production. The fourth generation encompasses genetically modified (GM) algae biomasses. There are several modern techniques that can help with this procedure some of which are, geo-synthesis, petroleum-hydro-processing, advanced biochemistry and various electrochemical processes. Through these methods, numerous algae traits can be optimized (Javed et al., 2019). The photosynthetic efficiency can be improved, light penetration can be increased and photoinhibition can be reduced. Like third-generation sources, there is no food-energy conflicts, undeveloped land can be used, biofuel conversion is relatively easy, various water sources can be used and with the enhanced ability of GM algae to sequester and assimilate atmospheric CO 2 , the detrimental effects of greenhouse gases can be alleviated by replacing fossil fuels (Abdullah et al., 2019). Though it is an enticing new source to exploit, It would be wise to prevent the neglect of the dangers that come with manufacturing such GM organisms (GMO). The possible leakage of GMO in pristine environments can dramatically alter the ecosystem both at a micro and macro level. This could shift the entire dynamic of the fauna and flora. This could lead to irreversible changes that can affect the downstream food web and cause concerning socio-economical changes in the long run. Moreover, producing biomass from GM algae on an industrial scale is still not feasible in a cost-effective and efficient way. The use of fourth generation feedstock can be very advantageous but caution and careful planning should prevail over the alluring benefits.

Thermophiles genome editing via CRISPR-Cas to enhance the biofuel production
Extreme thermophiles are still in the early phases of biotechnology and metabolic engineering. The genome editing of these microorganisms indicates the significant potential for industrial biofuel production. Despite being available in a sizable amount, there are a number of limitations on the utilization of thermophilic microbes in commercial synthesis of bioethanol from lignocellulosic substrate. Bioethanol was produced from lignocellulosic biomass using thermophilic bacteria because of their effectiveness, adaptability, and other desirable qualities including lower production costs, structural changes in cellulose at higher temperatures that make easy breakdown possible, and improved stability and targeted enzyme activity. Commercial lignocellulosic bioethanol production has a variety of challenges that prevent the widespread use of thermophilic microorganisms, despite their abundant availability. For a variety of reasons, thermophilic bacteria were ideally suited for the valorization of lignocellulosic biomass into bioethanol. Genome alterations of thermostable microorganisms for the aforementioned traits are exceedingly labor-intensive, rather than modifying microbes already employed in production of bioethanol for boosting thermostability. The ability to withstand high temperatures is a multigene characteristic. Manufacturing bioethanol often involves the use of high temperatures, which inhibits the multiplication of ethanolproducing microbes. Accumulation of HSP (heat shock proteins) and trehalose has been correlated to enhanced thermotolerance. Using possible microorganisms from the lab and the environment, CRISPR/ Cas9 may potentially be used to modify proteins implicated in thermoresistance (Figs. 3 and 4). S. cerevisiae and Zymomonas mobilis were able to tolerate higher temperatures and generate more ethanol after alteration of one amino acid in the NADH dehydrogenase and pyruvate kinase. Similarly, deletion of the gene encoding the transmembrane protein Dfg5 glycosyl phosphatidylinositol in S. cerevisiae resulted in increased thermostability (Sánchez-Muñoz et al., 2022). Using genome editing technologies, individual amino acid modifications can affect the specifics of cellulases. Previous difficulties with protein methodologies made it difficult to implement these modifications. In addition, these modifications were only produced in laboratory strains, making it impossible to implement them in commercial strains that produce bioethanol. Genome engineering through recombination can simplify the process of making targeted alterations to the DNA of microorganisms. These techniques can be used to alter the selectivity of cellulases during the synthesis of bioethanol (Ulaganathan et al., 2017). The potential of the cellulases to withstand heat was improved in a variety of microorganisms, including Bacillus sp., Clostridium thermocellum C. cellulovorans, C. phytofermentans, and Humicola insolens (Javed et al., 2019). Hemicellulases, which contribute to the formation of cell walls, can be used to produce ethanol since they have high pentose sugar content (Robak and Balcerek, 2018). The most well-known hemicellulase is endo-1, 4-xylanase, which randomly hydrolyzes the β-1, 4-xylosidic links in xylan to generate xylo-oligosaccharides (Balderas Hernández et al., 2021). Microbial xylanases from Yarrowia lipolytica, B. subtilis, Aspergillus usamii and A. niger contain amino acid changes that have boosted their capacity for thermotolerance. Similar to this, Thermomyces lanuginosus and Geobacillus stearothermophilus xylanases have been shown to have improved activity when certain amino acids get altered. (Ulaganathan et al., 2017). Genome editing methods based on CRISPR/cas9 can be used successfully and effectively to change hydrolytic enzymes to have desired properties.

Cadicellulosiruptor bescii
C. bescii is a viable option for fermenting sugars from crop leftovers into fuels since its optimal growth temperature is 78 o C. C. bescii might use a diverse range of cellulosic biomass (Brunecky et al., 2018). Since this organism's genome editing method is dependent on electroporation and uracil/5-FOA, the activity of restriction enzymes poses a challenge. Thus, either enzymatically inactive strains or transformation constructs that have been methylated are required. The multi-modular cellulases that are targeted for improving degradation of plant biomass are secreted by hemicellulases connected with the cell at the SLH (S-Layer Homology) modular domain and in the GDL (Glucan Degradation Locus) genomic region (Laemthong et al., 2022). Lignocellulose breakdown was not improved by the addition of exogenous cellulase (Xia et al., 2020). Overexpression of tapirins genes in C. bescii has the potential to enhance ligno-cellulose breakdown. Tapirins are unique substrate-binding proteins that have been discovered in the bacterium . By heterologously expressing the AdhE gene from Clostridium thermocellum, C. bescii was able to manufacture 0.6 g/L of ethanol from switchgrass (Williams-Rhaesa et al., 2018). When C. bescii was co-expressed with AdhE and ferredoxin NAD oxidoreductase, the ethanol titer for growth on crystalline cellulose was raised to 3.5 g/L at 60 o C. Although this is a significant advance in terms of titer, the byproducts acetoin and pyruvate suggest that more stable biocatalysts and genetic engineering are required to enhance electron flow and route carbon to the appropriate spot.

Thermococcus kodakarensis
T. Kodakarensis has temperature optimum for optimal growth is 85 o C and produced hydrogen gas by growing on starch, polysaccharide, and pyruvate (Crosby et al., 2019). Metabolically engineered T. kodakarensis produced higher hydrogen than the wild type and the engineered strain produced several times more hydrogen (Cao et al., 2022). Recently, by increased transcription of the genes involved in endogenous chitinase, N, N-diacetyl chitobiose catabolic pathway, H 2 production was achieved using chitin as a carbon source. Further improvement is achieved by the disruption of a glycolytic repressor and  selection for enhanced growth on chitin (Crosby et al., 2019). Recombinant protein translation of ORF-2 (Open reading frame) coding for the glucosamine kinase provided a more representation of how chitin enters central metabolism (Aslam et al., 2018). While these, H 2 -producing strains have been used for the production of industrial H 2 (4 mol H 2 /mol glucose), from the renewable abundant carbon source. T. kodakarensis from S. acidocaldarius express isoprenoid phytoene that has applications in the industrial manufacturing of biofuels (Ramu et al., 2021). The titers of phytoene synthesis improved by supplementing phytoene synthase and mutating the gene coding for acetyl CoA synthase. Further, many efforts are ongoing to improve hydrogen production and T. kodakarensis shows a good platform for bio-hydrogen production (Pfeifer et al., 2021). The CRISPR system has been used to design T. kodakarensis strains to target specific stretches of foreign DNA, which may provide a means of safeguarding industrial strains from viral contamination. (Akram et al., 2022).

Pyrococcus furiosus
With the help of the naturally competent uracil auxotrophic COM1 parent strain, P. furiosus has been metabolically engineered to produce ethanol, n-butanol, acetoin, and 3-hydroxypropionate. One of the biggest obstacles in using P. furiosus as a metabolic engineering host is its optimal growth temperature of 100 o C. Growing dense populations of cells at the optimum growth temperature allows for the efficient recruitment of heterologous enzymes with optimal activity near 100 o C; this problem can then be solved by lowering the temperature to the optimum range for the recombinant enzymes, as P. furiosus can maintain metabolic activity at temperatures as low as 70 o C, and the enzyme involved in product formation is active at this temperature. It is necessary to insert a heterologous pathway from a less thermophilic organism into P. furiosus because archaea predominate at higher temperatures and thermophiles rarely produce alcohol naturally . In the first demonstration of genetic engineering in P. furiosus, two soluble hydrogenases were knocked out, and the ability to use chitin was recovered by deleting a single base. Despite low titers, recent research has shown that heterologous expression of the bifunctional alcohol dehydrogenase (AdhE) from thermophilic bacteria can be used to produce ethanol. In addition, ethanol was produced by recombinant expression of AdhA from Thermoanaerobacter X514 and the native AOR (aldehyde oxidoreductase) of P. furiosus at 70 degrees Celsius via ferredoxin-dependent acetate reduction. Results showed that deleting the native AOR and expressing AdhA and AdhE decreased ethanol titer. It was found in this research that the AOR-AdhA pathway is more effective because P. furiosus is able to control its reducing equivalents. Additionally, by adding a lactate dehydrogenase under the control of PcipA, a titer of 0.3 g/L was achieved in production (Basen et al., 2012), and by adding a single enzyme in combination with the native enzyme in P. furiosus, titers of 2 g/L were achieved in ethanol production via a novel pathway. Yield of titers is 0.05 g/L at 72 o C when the carbon fixation cycle pathway is heterologously expressed in P. furiosus, resulting in the industrially important production of 3-hydroxypropionate. Using the same methods, Thorgersen et al. (2014) were able to increase the yield of 3-hydroxy propionate to 0.3 g/L by expressing accessory enzymes and optimizing growth conditions. Similarly, Keller et al. (2015) expressed six genes from three thermophilic bacteria for butanol production at 60 o C, but the yield of titers remained low (0.07 g/L). To produce H 2 from formate, P. furiosus carries a Thermococcus onnurineus formate dehydrogenase operon containing 18 genes; in addition, P. furiosus has an operon encoding a carbon monoxide dehydrogenase complex, which allows it to use carbon monoxide as a reductant. Genetic engineering for biofuel production is becoming more common, and P. furiosus of extreme thermophiles is the most impressive example of metabolic engineering to date.

Thermotoga maritima
Thermotoga maritima is an anaerobic, hyperthermophilic bacteria that thrive at 80 degrees Celsius and uses both simple and complex sugars to create H 2 at nearly the thermodynamic limit. Hydrogen generation in T. maritima, however, may be facilitated via genetic modification methods (Singh et al., 2018). Hydrogen generation was boosted, while growth and sugar intake were reduced, thanks to gene editing that disrupted lactate dehydrogenase. This is probably because of the abundance of electron carriers available through the aerobic hexose monophosphate route for hydrogen generation, whereas the disruption of an ABC maltose transporter, as shown by the sequencing of the whole genome, is the primary culprit of growth problems (Singh et al., 2018). Cellulases with a native signal peptide were expressed in a recombinant form in Thermotoga, and their cellulolytic activity was detected in the supernatant.

Thermus thermophilus
Thermus thermophilus, a kind of aerobic bacteria initially discovered in a Japanese hot spring, requires temperatures between 70 and 80 degrees Celsius to thrive (Ebaid et al., 2019;Wang et al., 2022a). Strain HB8 uses a denitrification process to grow anaerobically on sugars, lipids, peptides, and triglycerides (Smolinski et al., 2020). T. thermophilus takes longer than E. coli to develop a similar amount of biomass on a minimum glucose medium (Clément et al., 2021). Denitrification allows T. thermophilus to develop anaerobically, which is why metabolic engineering of this organism has been described. According to Wang et al. (2019), the heat-stable β-galactosidase gene TTP0042 serves as the foundation of the T. thermophilus gene system. Additionally, by altering the genome, they created an engineered strain with a high capacity for superoxide dismutases.

Sulfolobus sp
Sulfolobus species can develop at low Ph without being repressed, and it utilizes carbon sources for growth (Ebaid et al., 2019). Some species of Sulfolobus may thrive in sulfur environments, leading to the formation of sulfuric acid (Quehenberger et al., 2017). There is a requirement for further research of Sulfolobus metabolic engineering platforms, despite the fact that Sulfolobus sp. application is limited for industrial uses due to poor yields and long time for manufacturing (Quehenberger et al., 2017). Sulfolobus and S. sulfataricus strains were found to overproduce endoglucanase, proving that they could utilize cellulose effectively in the creation of new compounds (Girfoglio et al., 2012). Even while the evolution of the genetic system in Sulfolobus has been going on for quite some time, and there are numerous other examples of translation of proteins, none of them can be produced functionally.
White-rot fungi, including Pleurotus florida, Ceriporiopsis subvermispora, and Phanerochaete chrysosporium, have significant potential in CRISPR-Cas genome editing for biofuel production. By selectively degrading lignin in plant material, these fungi can enhance the efficiency of biofuel production. However, there is a risk of sugar losses during this process, which must be taken into consideration when optimizing the use of these fungi (Banu et al., 2021). Aspergillus terreus is a filamentous fungus that can be used for pretreatment in biofuel production. This fungus is known for its ability to delignify and degrade cellulose, which can enhance the efficiency of biofuel production . Trichoderma reesei is a soft rot fungus that can be used for detoxification in biofuel production. According to the findings of a study this fungus has the capability to eliminate weak acids, phenolics, and furans from plant tissue . As a consequence, the quality of the biofuel that is produced may be enhanced. The hydrolysis of filamentous fungi such as Trichoderma reesei, Humicola insolens, Termomonospora fusca, Aspergillus niger, and T. longibrachiatum may be utilized to produce biofuels (Noman et al., 2021). The capacity of these fungi to create xylanase, an enzyme that can digest the complex polysaccharide xylan found in plant material, makes it easier to convert that material into biofuels. Xylanase is an enzyme that can digest the complex polysaccharide xylan found in plant material. An enzyme known as xylanase has the ability to break down the xylan that is contained in plant matter. In general, the use of these fungi offers a significant lot of promise for enhancing the efficiency and sustainability of the production of biofuels. This is because fungi are able to break down complex organic materials into simpler components.
Spirogyra is one kind of algae that continues to improve as a biofuel feedstock. Genome editing has the potential to be a helpful resource here by boosting the productivity of algae farms that produce biofuel. Hydrolysis of Spirogyra hyalina was studied (Sulfahri et al., 2016) to see how it affects sugar yields. The hydrolysis procedure was performed using a range of heating times and enzyme types. The purpose of this research was to use Zymomonas mobilis to ferment Spirogyra hyalina into ethanol and to analyse the impact that varying the fermentation time and adding nutrients had on microbial biomass, pH shifts, reduced sugar, and ethanol yields. It has been shown that the sugar content of S. hyalina algae changes throughout the hydrolysis process, with the highest sugar content being produced by a mixture of α-amylase and β-amylase enzymes. The fermentation period and nutritional composition had an effect on the microbial biomass, pH, reducing sugar, and ethanol yield generated by the fermentation of the algae Spirogyra using Zymomonas mobilis. Algae biofuel production may be improved by the application of genome editing to enhance the hydrolysis and fermentation stages. By increasing the expression of genes involved in sugar metabolism and fermentation pathways, for instance, CRISPR-Cas genome editing can be utilized to improve the productivity of algae as a source of biofuel. Algae's resilience to environmental challenges like high salt or temperature can be enhanced by genome editing, thus contributing to the long-term viability of algae-based biofuel production.

Ways to enhance the biofuel production via CRISPR technology
Pretreatment, hydrolysis, and fermentation are the three phases that are involved in bioconversion of feedstocks into biofuels. The pretreatment process is the most significant, challenging, and expensive stage in turning biomass into ethanol. The four pretreatment process types-physical, physiochemical, solvent, and biological are most frequently used to break down cell walls so that cellulose and hemicellulose may be processed further. For greater effectiveness, all pretreatment techniques are often used in concert. The feedstock is then hydrolyzed using an acidic or enzymatic process after the pretreatment. The complex sugars in the source material are transformed into fermentable sugars during the hydrolysis process. Finally, monomeric sugars are transformed into ethanol or other alcohols through microbial fermentation. Four process topologies, including separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF), and consolidated bioprocessing (CBP), have been designed for the manufacture of biofuels (Fig. 5).

Separate hydrolysis and fermentation
The first method employed for the saccharification (hydrolysis) and fermentation of monomeric sugars is Separate Hydrolysis and Fermentation (SHF). As the name suggests, in this reaction hydrolysis and fermentation take place separately or sequentially. Even though it is inexpensive, quick, simple and can be optimized at either step, the endproducts can lead to the inhibition of the reaction and contamination is also quite prevalent. These drawbacks can decrease alcohol yields to noticeable levels. To circumvent this problem, hydrolytic enzymes can be added continuously through the reaction, process but this will diminish the cost-effective attribute of the method (Saini et al., 2015). To counter these drawbacks, an alternative method was developed called simultaneous saccharification and fermentation (Javed et al., 2019).

Simultaneous saccharification and fermentation
Simultaneous Saccharification and Fermentation (SSF) was developed to avoid the enzyme inhibition limitation of SHF. This is managed by coupling the hydrolysis and fermentation processes in the same vessel. SSF has shown to increase production of bioethanol by enhancing the hydrolysis rate, decreases contamination rates as well as decreasing enzyme inhibition by decreased enzyme loading (Erdei et al., 2012). Furthermore, due to the simultaneous reaction, microbial sugar consumption occurs immediately leading to low sugar concentration in fermenters (Saini et al., 2015). One main disadvantage of this process however, is the varying optimum temperatures for hydrolysis and fermentation. This could be overcome by using high temperature thermotolerant microorganisms (Javed et al., 2019).

Separate hydrolysis and Co-fermentation (SHCF) & simultaneous saccharification and Co-fermentation (SSCF)
In the previous methods, hexose sugars (glucose) are primarily Fig. 6. Schematic depiction of the application of CRISPR/Cas9, a genome editing method, in microbial cells to increase biofuel production. Cas9 may be delivered into microorganisms via techniques like the biolistic gene gun, electroporation, and heat shock transformation, with the sgRNA directing the delivery to the desired chromosomal location. Expression of Cas9 is controlled by the T7 promoter in the cassette, whereas expression of gRNA is directed by the U6 promoter in the scaffold. When a sgRNA hybridises with a target sequence, Cas9 helps separate the DNA strands, allowing for breakdown of the target gene's DNA. DNA is introduced into the active site of Cas9 in the correct orientation relative to a PAM binding site, enabling the Cas9 nuclease regions to possibly break the target DNA sequence 3 bp upstream of the PAM site. Two different processes, homology-directed repair and the more errorprone non-homologous end-joining (NHEJ), are used to fix DNA breaks (HDR). One-third of the time when a deletion or insertion occurs in the coding section of a gene, gene function can be recovered by resetting the correct reading frame. The transcription profile and kinds of alterations inside the targeted gene can be studied using real-time polymerase chain reaction (PCR), sanger sequencing, or nextgeneration sequencing.
converted into bioethanol and other alcohols. Wild-type Saccharomyces cerevisiae, is the most common fermenter used due to its high yield of ethanol, specificity and bioethanol and by-product tolerance. They are able to metabolize hexoses such as glucose but not pentoses like xylose.
Since quite a few biomass types have a significant amounts of hemicelluloses, fermenting pentose sugars would also be an effective mechanism for alcohol production. SHCF and SSCF were developed to tackle this issue and lead to a coupled fermentation of both pentose and hexose sugars (Erdei et al., 2012). The identification and selection of a single microbe that can efficiently lead to the targeted co-fermentation has proven to be very difficult due to the operating temperature and Ph levels (Saini et al., 2015). Instead of using one, a mixed-culture of both C6-fermenting and C5-fermenting microbes can be utilized. By choosing the right microbes and adjusting them to the required parameters of the reaction can lead to the conversion of all monomeric sugars in a synergistic fashion (Javed et al., 2019).

Consolidated bioprocessing
Consolidated Bioprocessing (CBP) or Direct Microbial Conversion (DMC), is the final method used for hydrolysis and fermentation. The key aspect of this configuration is the utilization of a single microorganism or microbial community for the production of all required enzymes and end-product biofuel, all in one reaction chamber. This method is favored because of its overall low cost in the components used (Saini et al., 2015). Unfortunately, ethanol and other alcohol yields were not as high as the previous configurations and the total time required for the completion of fermentation is more than 3-4 days, sometimes reaching up to 12 days. Unless these drawbacks are tackled, this mechanism will remain inefficient (Javed et al., 2019).
Originally designed to protect prokaryotes against phage assault, the CRISPR/Cas9 system is now often employed for site-specific gene editing. The short spacer DNA segments that border the very short repetitive DNA sequences make up the CRISPR. Spacer DNA, originally from a bacteriophage or plasmid, becomes part of the bacterial genome after interacting with a bacteriophage or plasmid (Fig. 6). Genes associated with CRISPR, known as Cas, code for proteins that may unwind or cut DNA, respectively. The bacterial CRISPR system recognizes a phage or plasmid by analyzing the sequences of the RNA it produces in response to it, and then directs the Cas enzyme to cleave the DNA at a predetermined site. Cas9, a cutting enzyme, may break the DNA at two active sites, one on each strand of the double helix. The bacterium Streptococcus pyogenes was the original host for this gene (Gostimskaya, 2022). The research indicates that bacteria protect themselves from phage attacks by encoding spacer sequences and palindromic repeats into a long RNA molecule, which is subsequently cleaved into pieces (called crRNAs) by the proteins Cas9 and trans-activating RNA (tracrRNA) (Ferreira and Choupina, 2022). It offers selectivity through CRISPR RNA (crRNA) and the transactivator CRISPR crRNA (tracrRNA). The Protospacer Adjacent Motif (PAM) is yet another important component in CRISPR-Cas that helps in effective binding to genomic DNA, placed right after the target sequence. The Cas9 can break two DNA helix strands around 3-4 nucleotides of the PAM's upstream region thanks to the sgRNA/Cas complex's 22 binding to the chromosomal target gene. As a result, a double helix breaks (Nor Azizah Ab Halim et al., 2022), which might be repaired using either non-homologous end joining (NHEJ) which is a DNA repair mechanism that directly results in insertion/deletion, but the homology-directed repair (HDR) approach demands the existence of homology-directed repair (HDR) enzyme, DNA template, and sgRNA (Fig. 7). A single guide RNA (sgRNA) derived from tracrRNA and crRNA was later shown to be an effective method for locating and cleaving certain DNA sequences using the Cas9 nuclease. The tracrRNA and crRNA bind to form a structure that resembles a hairpin loop, which the Cas9 enzyme uses to cleave DNA by identifying the crRNA as a guide. The cell may use either homology-directed repair (HDR) or non-homologous end-joining (NHEJ) to fix the damaged DNA. A DNA repair template is used to insert a particular DNA sequence for this purpose (Schiermeyer et al., 2019). In CRISPR/Cas9-based genome editing, transfection of target cells using a plasmid-or virus-based vector is a typical procedure. Using the sgRNA, Cas9 is instructed to preferentially damage certain regions of the genome. The DNA repair template is made to overlap with the sequences on each side of the cleft in order to further guarantee that the correct function is encoded. Site-directed mutagenesis has been used in theoretical and practical studies since its commencement. It has the potential to serve as a cutting-edge technique for changing the genomes of microbial cells to increase total biofuel production.
Using CRISPR/Cas9 to engineer bacteria to express desirable traits, such as b-cyclodextrin glycosyltransferase and spore-formation resistance in Bacillus subtilis, has been shown to be effective in a number of investigations. To enhance mevalonate or (R-R)− 2,3-butanediol synthesis as well as xylose consumption, the genome of S. cerevisiae was altered in a manner similar to that described above. According to Bae et al. (2021), it is anticipated that when additional CRISPR/Cas9 approaches are published, the use of this technology will progressively become a common component of the work done in laboratories. Electroporation, a biolistic gene cannon, or heat shock transformation are three methods that may be used to transfer Cas9 into microbial cells. Targeting certain sections of the genome requires the use of small guide RNAs, also known as sgRNAs. While the T7 promoter is responsible for activating the gRNA scaffold, it is the T7 promoter in a cassette that is responsible for Cas9 gene expression. Cas9 is able to increase the distance between the two strands of DNA and makes it possible for a sgRNA to hybridise with a particular site inside a targeted gene, which ultimately makes it easier to perform targeted DNA cleavage. Because the CRISPR/Cas9 system inserts the DNA into the active site of Cas9 in the precise orientation in relation to a PAM binding site (Fig. 7), the Cas9 nuclease domains are able to cleave the target DNA sequence three base pairs upstream of the PAM site. This allows the Cas9 nuclease domains to cleave the target DNA sequence. Approximately one-third of deletions or insertions in the coding sequence of a gene that repair the reading frame may restore gene activity. The real-time polymerase chain reaction (PCR), sanger sequencing, or next-generation sequencing are three methods that may be used to get information on the expression pattern of the gene as well as the sorts of mutations that are present.
A current CRISPR-Cas toolbox is inspired just by bacterial and archaeal defense system, which is employed to get rid of intrusive genes or bacteriophage DNA. The CRISPR-Cas mechanism is found in many different bacterial strains. To date, through the CRISPRdb internet database, 2762 genomes have already been examined, along with bacterial and archaebacterial strains (1302) with recognized CRISPR arrays found in the database that demonstrates the wide application of the CRISPR-Cas mechanism in prokaryotes. These systems can be customized for the manufacture of biofuel among a variety of traditional and pro-host bacteria owing to the abundance of diverse forms of CRISPR-Cas gear with variable capabilities.
Clostridium had previously been used to produce types of alcohol on a massive scale. The industrial synthesis of these alcohols has been hampered by its complicated genetic configurations and the absence of effective genetic tools for incorporating new genetic alterations, causing it to lag in the context of E. coli. Zhou et al. (2021) collected the early results on CRISPR-Cas-based genetic alterations in Clostridium species in a brief report. Owing to reduced crossover efficacy, mortality of Cas9 early translation, and vector integration events, CRISPR-Cas genetic engineering in Clostridium previously led to reduced transforming accuracy with fewer or even zero transformants, offering a significant challenge to the CRISPR-Cas system. These difficulties, however, were addressed by adopting vector-borne manipulating DNA to substitute its straight template, and the mortality of initial Cas9 expression was reduced by using inducible promoters (Bayro-Kaiser and Nelson, 2021). To increase butanol synthesis and specificity, a CRISPR-Cas9 gene editing method for excess butanol-generating C. saccharoperbutylacetonicum N1-4 was devised (Vamsi Krishna et al., 2022). For targeted gene editing of the butyrate kinase (buk) loci for butyrate generation and phosphotransacetylase (pta) loci for acetate synthesis, as solo and double variants, a CRISPR-Cas9 method originally proposed for C. beijerinckii has been implemented and evaluated in C. saccharoperbutylacetonicum. To do this, the single guided RNA (sgRNA) was generated utilizing a short RNA promoter (PsRNA) from C. beijerinckii and transcription of the CRISPR-associated open reading frame (ORF) was carried out downstream of the lactose inducible promoter (PbgaL) in Streptococcus pyogenes. Table 1.

CRISPR-based molecular networks control gene transcription, resulting in improved biofuel production
Generating genetically engineered microbes having acceptable gene mutations for the synthesis of molecules and biofuel typically needs a lengthy and intense strain improvement strategy due to intricate biochemical control systems (Ko et al., 2020). But even so, the constructed species may have a low performance of intended bio-products caused by genetic strain coupled with rising transcription of mutant genes that need further inducers or thermal fermentation limitations for controlled or reliable transcription (Peng et al., 2022). Old methods may also cause disturbances in the equilibrium of biochemical activity and quantity of key intermediates at various phases of processing, resulting in a micro yield and lowering the system's commercial feasibility (Jacob et al., 2021). Consequently, an auto-induction system can be created to switch on/off the genetic circuit to regulate the phenotypic expression in response to stimuli. The designing of such genetic circuits is a hurdle because the regulators which may regulate the expression of many genes are scarce and the methods to manipulate the target genes still need to be developed (Das and Chatterjee, 2019). Foreign input signals may initiate gene regulation, which in turn triggers a cascade in the inner circuit design, where engineered sgRNA and Cas9 promoters mediate programmable gene repressions that lower endogenous expression of genes to boost the generation of biofuel. The nuclease-deficient Cas9 protein (dCas9) has been described to be utilized in conjunction with sgRNA to target gene promoters, resulting in transcriptional repression (CRISPRi). The CRISPR method has been shown to lag behind the use of CRISPRi in bacteria because transcriptional activation domains are few in prokaryotes (Yao et al., 2018). Yet, with the continual discovery of new and more potent transcription regions, gene activation should continue to improve. Furthermore, each genetic component's expression level may be adjusted to perfect CRISPR-based gene repression/inactivation. Increasing the expression level of dCas9 or sgRNA, for example, significantly increased transcriptional inhibition (Zhan et al., 2020).
Single or multiple genes can be silenced using sgRNA and tracrRNA in the CRISPR locus. Multiplex gene repression can be carried out through the CRISPR Type I system. The metabolic flux can be redirected by shutting down the competing pathways through CRISPRi-mediated inhibition in E. coli BW25113 to increase n-butanol yield (Zhao et al., 2021). The available quantity of NADH and acetyl CoA can be limited by catalyzing the reaction to convert acetyl CoA into acetate, succinate, lactate, and ethanol using various E. coli strains like AtoB, Crt, AdhE2, Hbd, and Ter (Abdelaal et al., 2019). Consolidated bioprocessing (CBP), which combines substrate hydrolysis with alcohol fermentation using a microbial consortium to produce a one-step conversion process (Singhania et al., 2022), is another prominent use of CRISPR-Cas9 for increasing the production of alcohol. Gene ack coding for acetate kinase and ldh coding for lactate dehydrogenase can be knocked down to divert the C-flow towards the solvent production pathway (Ceron-Chafla et al., 2022). The expression of butyrate kinase can lead to an increase in the butanol yield up to 6.65 g/L in E. coli strain 734B (Shanmugam et al., 2020). Similarly, the CRISPRi mechanism can be utilized for downregulating the expression of enzyme hydrogenase to increase bioethanol manufacturing. Genes like xylT (xylose symporter), ctfA/B (CoA-transferase) and xylR (xylose regulator) can be upregulated and resulted in the production of 11.5 g/L butanol, 4.25 g/L acetone, and 6.37 g/L ethanol in strain NCIMB 8052. The outcome showed an 87.2% improvement as compared to untreated output (Wen et al., 2017). By knocking down the transcription of relevant genes selectively, Guo et al.
(2022) employed CRISPR and CRISPRi systems to regulate the 1,4-butanediol (1,4-BDO) biosynthetic pathway and reroute C-flow in an E. coli strain. Two large pathway gene cassettes (6.0 and 6.3 kb in length) encoding six genes were combined using CRISPR-Cas9 technology: sucD, cat1, cat2, 4hbd and bld from Porphyromonas gingivalis, C. kluyveri, P. gingivali, and C. saccharoperbutylacetonicum respectively (Yoo et al., 2022). It has been demonstrated that is a flexible technique for lowering endogenous gene expression without compromising gene function, allowing metabolic flow to be fine-tuned toward the desired products.

Multiplex automated genome engineering (MAGE) technology using CRISPR for biofuel production
The restricting factor in achieving the desired trait with exceptional biological makeup, such as rising bioenergy efficiency, is the manufacturing of an appropriate collection of options with desired gene mutation, guided by a time-consuming assessment method of gathering unique optimistic recombinant plasmid from the main channel of unedited origins (Adegboye et al., 2021). Conventional biotechnology typically impacts one chromosomal region in a cycle, demands a great deal of time and work, and only a few primer pairs with poor mutation rates were produced as a consequence (Volke et al., 2022).
With the invention of the CRISPR-Cas genetic modification method, the editing effectiveness was greatly improved with the precise chromosomal alteration, highly boosting the prospects for varied gene engineering of target DNA in numerous loci with only one cycle of mutations. The multiplex automated genome engineering (MAGE) strategy, which is based on designing widely varying mutants with various modifications per genetic trait, has a high potential for creating an array of genetic differences in key genes while keeping other genes unaltered, and it can easily avoid the evaluation and selection of altered variants (Wannier et al., 2021). The MAGE system was effectively used to increase lycopene synthesis by around five times in E. coli by enhancing the 1-deoxy-D-xylulose-5-281 phosphate (DXP) biogenesis (Park et al., 2018). The MAGE technique can now create increased genetic variety in bacteria, possibly permitting the building of organic biofuel pathways, thanks to the rapid progress of the Cas9-based system. In bacterial genomes, MAGE regulated by nucleases has been exploited. To enhance the yield of isopropanol production using E. coli, Park et al. (2018) developed CREATE (CRISPR Enabled Trackable Genome Engineering), a revolutionary multiplex genome engineering approach that integrates MAGE with CRISPR-Cas9 and barcoding technique. E. coli mutant strain PA06 was designed to produce isopropanol at increased productivity of 0.40 g/L/h (yield of 0.62 mol/mol) primarily through codon modeling of five genes (adc, ctfAB, thl, adh, and atoDA), influenced the constitutive promoter J23119 in a plasmid with low copy numbers (Shanmugam et al., 2020).
Two-dimensional material called graphene is made of carbon atoms with sp 2 hybridization. In order to create hybrid and nanocomposites, several new switchable device technologies  combine stimulus-induced optical and electrical changes in 2D graphene and derivatives with nanomaterials. Stretching hydrogen bonds (H -) in precursor droplets between two substrates to form a liquid bridge is a straightforward and adaptable approach for synthesizing semiconductor nanomaterials (CdS and TiO 2 ) dispersed liquid crystals (NDLC) or graphene NDLC. There aren't many LCs with excellent classically oriented nematic phases. Flexible electronics, high-contrast smart displays, and opto-electronics have all been improved by thin-film nanocomposite materials and switchable technology. These advantages are enhanced by GDLC and NDLC composite sensors for chemical flammability, explosiveness, and toxicity. The inclusion of biopolymers, biosensing, and antimicrobial applications are proposed. Over many years, GDLC hybrid nanocomposite matrix was created for smart switchable devices.
Nanographene, with its one-of-a-kind features such as a large surface area and outstanding mechanical capabilities, has the potential to be utilised in composite or filler material applications within the biofuel industry. It is possible that the conductivity, stability, and catalytic activity of electrodes or catalysts used in biofuel cells or fuel synthesis operations might be improved by introducing nanographene into biofuel production systems (Pal et al., 2022). In addition, the photocatalytic characteristic of nanographene may be used to facilitate the effective transformation of solar radiation into usable energy, which might be of use in the manufacture of biofuels. It emphasizes the requirement for large-scale manufacture of high-quality graphene for its utilization in nanoelectronics. Because of its one-of-a-kind qualities, graphene has been successfully combined with a variety of other materials, including metals, metal oxides, and polymers, which has led to the development of graphene nanostructures and composites . The potential application of CRISPR-Cas technology to optimize biofuel production systems, build graphene-based catalysts, engineer biomass conversion pathways, and improve microbial ethanol production is the focus of the research on graphene and CRISPR-Cas genetic modification for biofuels. This combination of genetic engineering methods with cutting-edge materials, like as graphene, offers the potential to make significant contributions to the development of sustainable energy solutions and the advancement of the biofuels industry. The manufacturing of geopolymers that have been changed by magnetic nanoparticles have an exceptional oil sorption capacity due to the creation of pores inside the material (Silveira Maranhão et al., 2021). The gene editing technique known as CRISPR-Cas can be used to improve the characteristics of microorganisms that are engaged in the generation of biofuel. This might possibly lead to the development of microbes that are capable of effectively digesting and using oil spills. It is anticipated that it will be feasible to build novel and efficient methods for environmental cleanup of oil spills utilising biofuel production systems if geopolymer matrices are combined with genetically engineered microbes. Due to its simplicity, speed, and unprecedented resolution, the CRISPR-associated (CRISPR/Cas) adaptive immune system has been widely used for gene editing in bacterial and eukaryotic cells (Cho et al., 2018). The CRISPR interference (CRISPRi) approach, which regulates the target gene transiently or constitutively, avoids cell death through genome disruption. This review covers CRISPR/Cas genome editing in bacterial systems. Bacteria produce biochemical, biofuel, and medicinal products/precursors using CRISPR technology. We employ CRISPR/Cas and CRISPRi systems with synthetic pathways to boost productivity and yield/titer scan in industrially common bacteria like Escherichia coli.

Increased biofuel host specificity with endogenous CRISPR-Cas
The CRISPR-Cas9 method has also been employed to genetically modify established model bacteria like Clostridium acetobutylicum, C. beijerinckii, and many eccentric genera but with distinct traits, all of which were previously hampered by unresolvable DNA frameworks and a lack of efficient genetic tools, enabling for yet more value biofuels. Clostridium species can produce diverse alcohols via a variety of methods, including preferential sugar consumption, altering carbon flow, and using inducible promoters, as described in this paper. Recombinant protein Cas9 expression is extremely toxic to prokaryotic chromosomes, resulting in lethal DNA rupture and, consequently, poor transformation ability and genetic manipulation loss (Arroyo-Olarte et al., 2021). The growing availability of CRISPR-Cas technology for Clostridium could help to alleviate Cas9 toxicity and poor transformation ability (Riley and Guss, 2021).

Increased capacity to use substrates
Industrial Clostridium isolates that need little feedstock to yield higher alcohol fermentation have been developed, which is viewed as a big step forward in cutting manufacturing costs (Fackler et al., 2021). Carbon catabolite inhibition prevents Clostridium species from using other carbohydrates when glucose is present in the substrate. This can be overcome by altering genes involved in sugar absorption, allowing Clostridium species to use a wider spectrum of carbohydrates. Rapid carbon collection for bacterial fermentation before lignin incorporation has been considered a viable option. Acetogenic anaerobic bacteria may convert carbon derived from gaseous sources (Syngas -H 2 , CO, and CO 2 ) to liquid fuels via the Wood-Ljungdahl pathway (Benevenuti et al., 2021).

Improved solvent synthesis by redirecting metabolic flux
Aside from limiting competing routes, restoring equilibrium and directing carbon flux is thought to be some other effective strategy for increasing biobutanol generation in a microbiological system . An E. coli EMJ50 strain that has been designed for increased synthesis of the endogenous acetoacetyl-CoA thiolase (thl), formate dehydrogenase (fdh1), alcohol dehydrogenase (adhE2) from C. boidinii allowed for the production of biobutanol utilizing glucose (Banu et al., 2021).
C. acetobutylicum produces aldehyde dehydrogenase (adhE2), which is sensitive to oxygen. Bogorad et al. (2014) used enzymes from different bacteria, including CoA-acylating propionaldehyde dehydrogenase (PduP) from S. enterica, alcohol dehydrogenase (adhA) from L. lactis, and formate dehydrogenase (fdh1) from C. boidinii, to modify strain EMJ50. The modified strain was able to produce 0.82 g/L of butanol from glucose, with a yield of 0.068 g/g, under slightly oxygenated conditions. However, the butanol yield was slightly lower than under completely oxygen-free conditions, likely due to the production of citric acid from acetyl-CoA, which is also used as a precursor for butanol. By reducing the expression levels of the 5' untranslated region (UTR) of citrate synthase, carbon flow was switched to butanol synthesis.

Genome editing increases inhibitor tolerance
Genome editing aims to develop bacterial resistance to very damaging substances including fermentation process inhibitors and particular growth inhibitors. Physiochemical pre-treatment of lignocellulosic biomass produces several cellulolytic enzymes and microbial growth inhibitors during fermentation, lowering ethanol yields. These inhibitors fall into three categories: low-acid organic acids, furan derivatives, and phenolic chemicals. Inhibitor resistance in Saccharomyces cerevisiae and other ethanogenic bacteria has increased bioethanol output. Replacing one amino acid in four genes at various places increased constitutive acetic acid tolerance. Single amino acid modifications in multiple RpoD protein locations increased furfural resistance in Z. mobilis. Transposon insertion altering the SSK2 gene may increase furfural tolerance in S. cerevisiae. Thus, CRISPR/Cas9 might increase enzyme tolerance to inhibitors in microbes to promote biofuel production.

Genome editing for altering cellulases and hemicellulases
Genome editing tools allow for precise control over how amino acid substitutions influence cellulase activity. Problems with protein engineering techniques made it difficult to implement these modifications in the past. It was also challenging to duplicate these changes in commercial strains that produce bioethanol because they were only developed in the lab. Precision alterations to the genetic code of microorganisms may now be accomplished with greater ease thanks to genome engineering techniques based on recombination. During bioethanol production, these methods can be utilized to modify the selectivity of cellulases. Multiple microorganisms, including Cellulobacter cellulovorans, Bacillus sp., Humicola insolens, Cellulobacter phytofermentans, Cellulobacter thermocellum, and Melanocarpus albomyces have enhanced cellulase's thermal stability. Using a congo red plate test, Bacillus MSL2 was identified as a cellulase-secreting strain among 200 bacterial isolates gathered from Thai rice paddy (Sriariyanun et al., 2016). The best candidate for cellulase synthesis was MSL2, which exhibited the greatest global cellulase activity and the second-highest CMCase activity. The isolated enzyme was determined to be an endoglucanase after extensive testing. Kinetic characteristics and optimal circumstances for enzyme hydrolysis were also investigated. Because of its tolerance to IL, MSL2 may be used in a single-step, combined pretreatment and saccharification process; this has potential to advance the state of the art in lignocellulosic biorefinery technology.
Hemicellulases, which help create cell walls, can be converted into ethanol because of their high pentose sugar content. The most wellknown hemicellulose enzyme is endo-1, 4-xylanase, that functions to catalyse the breakdown of the β-1, 4-xylosidic links in xylan to create xylo-oligosaccharides. Prior literature on protein engineering suggests that modifying certain amino acids in xylanase enzymes can affect their substrate selectivity. Amino acid substitutions have improved the thermotolerance of xylanases from Yarrowia lipolytica, Aspergillus usamii, A. niger and B. subtilis. Similar improvements in activity have been shown for xylanases from Geobacillus stearothermophilus and Thermomyces lanuginosus both of which contain the amino acid lysine. Hydrolytic enzymes can be modified to have certain features by using CRISPR/cas9based genome editing techniques. Low-value carbons were used in an eco-friendly manner to produce fuel-like compounds and oleochemicals by metabolic engineering of Yarrowia lipolytica (Darvishi et al., 2017). The metabolic pathways of acyl-CoA and acyl-ACP were redirected to accomplish this. Overexpression of xylose dehydrogenase, heterologous xylose reductase and endogenous xylose kinase also enhanced capacity of Y. lipolytica to consume xylose. Steady development on xylose substrates was enhanced by overexpressing XKS and XDH. Sustainable production of high-value fuels and oleochemicals may be possible with the help of these genetic engineering strategies to boost lipid accumulation in Y. lipolytica. Thermostable versions of TLL, a lipase useful in the manufacture of biodiesel, were predicted using computational techniques by Zhu et al. (2022). The research team zeroed down on the impact of charged residue substitutions on TLL thermostability and settled on R209. Ten different TLL variations were created, and three of them (R209I, R209M and R209H) had improved reaction temperatures, melting points, and thermal stability. Molecular dynamics simulations showed that the enhanced thermostability of these mutants was due in large part to the stability of the 250-loop. This work demonstrates the use of computational techniques for enzyme design, and the R209 mutants that resulted show promise as industrial possibilities for biodiesel generation.

Off-target effects
The consequences of CRISPR-based gene therapy are considerable, especially when it comes to off-target outcomes. With microbial biotechnology, off-target impacts are less frequent, but they remain a concern. We also talk about the impacts of off-target effects on prokaryotic systems as a result. Contrary to ZFN and TALEN, Cas9 proteins may affect bacterial systems more so than eukaryotes. Researchers are increasingly using prokaryotes to make biofuels due to the smaller genome size, genetic diversity, and decreased proclivity for mutations caused by Cas9 .

Reduction of off-target effects by Cas9 modification
The temporal sequence and spatial regulation of Cas9 protein production are all important factors in the success of a CRISPR procedure. Continued Cas9 protein synthesis isn't always desirable, and it may happen if gRNA and Cas9 are produced by the same plasmid. However, because the targeted genes are essential for host cell survival, chronic Cas9 production might cause off-target effects and/or start a reaction to DNA damage (Zhang, 2021). To reduce Cas9 toxicity, researchers have used inducible promoters and transitory Cas9 expression. The "codon-optimized" method may be employed on the nucleotide makeup of the specific and target species to allow flawless Cas9 transcription for many microbial species (Xu et al., 2021). The FokI nuclease motif, which is found in both ZFNs and TALENs, is employed in an extremely efficient strategy for Cas9 alteration to reduce off-target effects (Vakulskas and Behlke, 2019). The higher specificity was due to FokI's tight heterodimer need, as opposed to Cas9's monomer, which resulted in better target binding. The CRISPR system is characterized as a "double nick" mechanism because it uses a Cas9 nuclease to produce two proximal, distinct strand nicks. Depending on the effect on the target genes, either NHEJ or HDR can be used to repair a double-nick-caused DSB (Bischoff et al., 2020). Uslu et al. (2021) created Cas9 (eSpCas9) and hypothesized that Cas9's propensity to unwind and rewind DNA in non-target locations was the source of off-target cleavage. A positive charge pocket in the non-target strand of the Cas9-gRNA and its target sequence from Streptococcus species was identified during crystallographic analysis of the Cas9-gRNA and its target DNA from Streptococcus species, which was later altered to get a low selectivity for Cas9. To summarize, most concerns concerning off-target CRISPR mutagenesis may be addressed by combining the correct Cas9 variant with an accurate and strategic gRNA design. Researchers have found that new restriction enzymes from Type V CRISPR-Cas systems, such as Cpf-1, offer advantages over Cas9 and eSpCas9 in addition to these differences (Shanmugam et al., 2020). Its ability to generate sticky edges having 4 or 5 bp overhangs, which simplifies NHEJ-mediated knock-in and its ability to cut DNA with the short a crRNA rather than the long tracrRNA are its two most significant features (Javed et al., 2019). Pre-crRNA digestion and multiplex genome editing are made possible by Cpf-1's induction of RNAse III activity in addition to DSBs, with little to no off-target consequences (Wawszczak et al., 2021). The efficiency of genetic alteration is significantly increased by Cpf1, the effector protein, which can distinguish T-rich PAM regions from G-rich PAM regions (Shanmugam et al., 2020). These families may be capable of gene knockdown. When more sophisticated sequencing techniques like GUIDE-Seq (Shanmugam et al., 2020) and Digenome Seq (Kim, 2018) are made available to labs all around the world, CRISPR technology will once again outperform the competition.

Using sgRNA to reduce off-target effects
In reality, multiple sgRNAs could produce the same target gene editing (TGE), but the secret to attaining a more effective TGE grade is picking a target gene without or with few genetically identical sequences close. Several algorithm-based processes, like CRISPR DESIGN (http ://crispr.mit.edu), E-CRISP (http://www.e-crisp.org/E-CRISP/reannot ate crispr.html), and CHOPCHOP (http://chopchop.cbu.uib.no) have been created based on varying degrees of dominance, sequence homology, quantity, and position . Manghwar et al. (2020) found a link between the gRNA and Cas9 and the number of off-target effects. It was discovered that shorter gRNAs of 17-18 nt had exceptionally low off-target impacts while retaining results that are on target, and that this was because smaller genomes had fewer "wrong" gRNA base-pairing target positions.

Regulation of byproducts in biofuel production
Industrial-scale fermentation seeks high productivity, yield, and product concentration. Alcohol dehydrogenase (ADH) and pyruvate are decarboxylated to acetaldehyde to produce ethanol in S. cerevisiae . Native S. cerevisiae generates glycerol and acetate while making bioethanol. Since carbohydrate feedstock costs the most, expanding sugar-based ethanol production is crucial. The metabolism of ethanol, glycerol, and acetate in S. cerevisiae is difficult because it involves several genes, including the GPD, ADH, and ALD genes. In two phases, NAD + -dependent glycerol-3-phosphate dehydrogenase (GPD) isoenzymes GPD1 and GPD2 reduce dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate. GPD2 activity maintains redox balance, whereas GPD1 activity reacts to osmotic stress. Glycerol-3-phosphate phosphatase (GPP) dephosphorylates G3P into glycerol. Glycerol may be produced from 4% of substrate carbon. Preventing this molecule's synthesis may increase ethanol production. Aldehyde dehydrogenases (ALDH) oxidise acetaldehyde to acetic acid, whereas alcohol dehydrogenases (ADHs) convert ethanol and acetaldehyde to NAD + or NADP + . The ADH2 gene expresses alcohol dehydrogenase II, which increases ethanol synthesis. It catalyses ethanol-acetaldehyde conversion. Acetate, a yeast byproduct, inhibits cell proliferation and ethanol production in S. cerevisiae. Two yeast subcellular locations create acetic acid. ALD4 and ALD5 encode mitochondrial ALDHs. ALD6, ALD2, and ALD3 encode NADP + -dependent cytosolic enzymes. Ald6p has been demonstrated to produce acetate during sugar fermentation, whereas Aldp4p may produce acetic acid. The ALD4 gene has been studied less than the ALD6 gene in ethanol metabolism pathway optimization. The excessive accumulation of glycerol and organic acids throughout the industrial ethanol production process led to a decrease in ethanol concentration. To increase ethanol production, GPD2, FPS1, and ADH2 were deleted from S. cerevisiae engineering strains using the CRISPR-Cas9 technique (Yang et al., 2022). RNA sequencing and transcriptome analysis were used to examine the effect of gene loss on gene expression. The results revealed that utilising 50 g/L of glucose as a substrate, the modified S. cerevisiae SCGFA produced 23.1 g/L ethanol, an increase of 0.18% over the wild-type strain. GPD2, FPS1, and ADH2 were concurrently deleted to achieve this. The SCGFA strain converted ethanol at a rate of 0.462 g per g of glucose. Glycerol, lactic acid, acetic acid, and succinic acid contents were also 22.7%, 12.7%, 8.1%, 19.9%, and 20.7% lower in SCGFA than in the wild-type strain. Glycolysis, fatty acid, and carbon metabolism may have an effect on the ethanol production of SCGFA, according to a KEGG research of the up-regulated gene enrichment. Thus, there is a lot of potential for the engineered strain SCGFA to produce bioethanol.

Effect of genome editing in microbes on environment and safety issues of biofuels
Genome editing in micro-organisms might boost biofuel output, but it poses environmental and safety concerns.
1. Increased Efficiency: CRISPR-Cas9 can change biofuel-producing bacterial genetic material. This improves metabolic pathways, making biomass-to-biofuel conversion more efficient. Genome editing increases biofuel productivity, making it more sustainable and profitable (Lu et al., 2022b). 2. Environmental Impact: Biofuel generation using genome-edited microorganisms relies on the alterations (Hemalatha et al., 2023). Released modified bacteria must be monitored for interactions with natural species. Before large-scale deployment, containment and risk evaluations may reduce ecological damage. 3. Genetic Stability: Microbial genome editing can make specific alterations, but their stability is a worry. Genetic recombination or other genetic processes may cause unintended genetic changes or characteristic loss. To keep biofuel-producing modified bacteria safe and efficient, genetic alterations must remain stable (Javed et al., 2019). 4. Biosafety criteria: Genome-edited microorganisms for biofuel generation must follow biosafety criteria (Xu et al., 2022). Before commercial release, changed microorganisms must be risk assessed for safety. Regulatory frameworks differ by country. 5. Ethical Considerations: Along with environmental and safety problems, consider ethics. These may include assessing the environmental impacts of releasing altered bacteria and resolving ethical issues surrounding live creature alteration (J. .
Genome editing and its uses are expanding. Responsible and sustainable genome editing in biofuel production requires ongoing study, stakeholder participation, and risk-benefit analysis.

Associated challenges for genome editing of thermophiles
Genome editing of thermophiles, high-temperature microorganisms, is more difficult than mesophilic species. Thermophile genome editing challenges: 1. Genetic Tools and Techniques: Thermophiles have complicated genetic systems, making genome editing difficult. Genome editing requires genetic engineering techniques including effective transformation procedures and gene delivery systems. 2. Thermophiles flourish at high temperatures, making genome editing challenging. Genome editing enzymes like restriction enzymes and polymerases may not be stable or functioning at thermophilic growth conditions. Creating and optimizing temperature-tolerant genome editing tools and enzymes is crucial. 3. DNA Repair systems: Thermophiles have powerful DNA repair systems to survive severe circumstances. Genome editing may be hindered by these repair pathways. Precision genome editing in thermophiles requires understanding and overcoming these repair processes . 4. Transformation Efficiency: Compared to mesophilic species, thermophiles may transfer foreign DNA less efficiently. Editing reagents and donor DNA are harder to distribute to thermophiles because to their reduced innate ability. Genome editing requires effective thermophile-specific transformation strategies. 5. Off-Target Effects: Genome editing may cause genetic alterations in unexpected places. Thermophiles have complicated genetic systems and little genome dynamics information, making off-target impacts harder to anticipate and identify. Minimizing off-target effects requires genomic alteration analysis and confirmation. 6. Genetic Diversity: Thermophiles have many strains and varieties.
Diversity affects genome editing effectiveness and stability. For consistent and trustworthy findings across strains, thermophile genetic diversity and genome editing must be understood.
These issues need molecular biology, bioinformatics, and thermophile-specific study. Technology and knowledge will improve genome editing techniques and tactics for thermophiles, allowing advances in bioenergy, bioremediation, and biotechnology.

Conclusion
To deal with the current problem of global energy demand, effective measures are required. The CRISPR-Cas gene editing technique can boost extremophile biofuel output. Traditional strain enhancement approaches are time-consuming and lack gene expression control. CRISPR-Cas makes genetically modified bacteria with desired gene alterations easier and more accurate. Inducible gene expression regulation allows CRISPR-Cas to fine-tune phenotypic expression in response to certain stimuli. Gene suppression or inactivation using CRISPRi redirects metabolic flow to biofuel production pathways. Nuclease-deficient Cas9 (dCas9) and tailored sgRNA allow transcriptional suppression, increasing gene regulation. Multiplex automated genome engineering (MAGE) allows extremophiles to simultaneously edit several genes, improving CRISPR-Cas gene editing. This method creates a wide set of genetic alterations in essential genes without time-consuming examination and selection of altered variants. CRISPR-Cas technology also addresses Cas9 toxicity and poor transformation capacity in bacteria like Clostridium species, enabling host-specific biofuel generation. Redirecting metabolic flow inhibits competing pathways and boosts biofuel production in certain strains. CRISPR-Cas genome editing may improve substrate utilisation, fermentation inhibitor tolerance, and biomass breakdown cellulases and hemicellulases. Extremophiles can convert more feedstocks into biofuels by carefully changing their genomes. CRISPR-Cas gene editing in extremophiles for biofuel generation might solve strain improvement, metabolic engineering, and substrate utilisation issues. CRISPR technology will improve, enabling more efficient and sustainable biofuel production. Efforts in the scientific community are currently focused on discovering the most effective way to improve the performance of biofuels derived from lignocellulosic materials while using as little water and electricity as possible. The optimization of pretreatment, hydrolysis, fermentation, and purification, all of which are necessary steps in the manufacturing of biofuels, must be taken into account in future research. Around one-third of the entire budget goes into the most expensive part of the process, pretreatment. To maximize product yield under adverse conditions, it is necessary to produce genetically modified microorganisms with the needed fermentative and cellulolytic capabilities and to employ increased co-cultures. Research into effective bioconversion technologies for biofuel production has received a lot of attention in recent years. Every method used to influence biofuel production comes with its own set of restrictions. Improving the output and quality of biofuels necessitates resolving these constraints as effectively as possible. Because of these restraints, research has focused on using genetically modified microbes to power the bioconversion of vast quantities of inexpensive raw materials into biofuels.
As demand for fossil fuels continues to outstrip supply, environmentalists have pushed scientists to develop more sustainable methods of producing energy. The purpose of this review was to shed light on current developments in CRISPR/Cas9-mediated genome engineering of microbial cells in the hopes of increasing the efficiency with which these cells produce biofuels. Genome editing using CRISPR/Cas9 could be used to redesign bacteria to increase product concentration, inhibitor tolerance, cellulase and hemi-cellulase modifications, product yield, and tolerance. By combining these methods, we can predict an increase in the quality of microbial cells that can produce commercial quantities of bio-fuels at a low cost.

Future perspectives
Research and technological advances will improve CRISPR-Cas technology's efficiency, specificity, and flexibility. Cas protein variants improve gene expression and regulation. Extremophile genome alteration will enhance biofuel production. CRISPR-Cas may help in regulation of metabolic pathways of extremophiles. Precision gene expression modulation optimises metabolic flow for biofuel production. Novel extremophilic micro-organisms may be identified with property of higher biofuel synthesis. CRISPR-Cas gene editing may improve biofuel host specificity. Gene manipulation may enhance these organisms' usage of lignocellulosic biomass or syngas. This increases biofuel feedstocks, efficiency, and sustainability. Lignocellulosic substance inhibits biofuel fermentation. CRISPR-Cas may help bacteria survive these inhibitors and convert biomass into biofuels. By altering inhibitor detoxification and resistance genes, researchers can boost biofuel production. Biofuel production is promising using CRISPR-Cas gene editing and synthetic biology. CRISPR-based genome editing and synthetic biology methods like constructing synthetic genetic circuits and pathways may enhance biofuel production and cell activity. Biofuel production innovation and sustainable bioenergy systems will result from this interdisciplinary approach. CRISPR-Cas gene editing might change extremophile biofuel production. This advanced technology optimises metabolic pathways, host specificity, and inhibitor tolerance. CRISPR-Cas and synthetic biology will accelerate sustainable biofuel production.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
No data was used for the research described in the article.