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Review

Microbial Genome Editing with CRISPR–Cas9: Recent Advances and Emerging Applications Across Sectors

by
Chhavi Dudeja
1,
Amish Mishra
1,
Ansha Ali
1,
Prem Pratap Singh
2,* and
Atul Kumar Jaiswal
1,*
1
Jamia Hamdard, School of Engineering Sciences & Technology, New Delhi 110062, India
2
Department of Viticulture & Enology, University of California, Davis, CA 95616, USA
*
Authors to whom correspondence should be addressed.
Fermentation 2025, 11(7), 410; https://doi.org/10.3390/fermentation11070410
Submission received: 14 June 2025 / Revised: 9 July 2025 / Accepted: 11 July 2025 / Published: 16 July 2025
(This article belongs to the Section Fermentation Process Design)
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Abstract

CRISPR technology, which is derived from the bacterial adaptive immune system, has transformed traditional genetic engineering techniques, made strain engineering significantly easier, and become a very versatile genome editing system that allows for precise, programmable modifications to a wide range of microbial genomes. The economies of fermentation-based manufacturing are changing because of its quick acceptance in both academic and industry labs. CRISPR processes have been used to modify industrially significant bacteria, including the lactic acid producers, Clostridium spp., Escherichia coli, and Corynebacterium glutamicum, in order to increase the yields of bioethanol, butanol, succinic acid, acetone, and polyhydroxyalkanoate precursors. CRISPR-mediated promoter engineering and single-step multiplex editing have improved inhibitor tolerance, raised ethanol titers, and allowed for the de novo synthesis of terpenoids, flavonoids, and recombinant vaccines in yeasts, especially Saccharomyces cerevisiae and emerging non-conventional species. While enzyme and biopharmaceutical manufacturing use CRISPR for quick strain optimization and glyco-engineering, food and beverage fermentations benefit from starter-culture customization for aroma, texture, and probiotic functionality. Off-target effects, cytotoxicity linked to Cas9, inefficient delivery in specific microorganisms, and regulatory ambiguities in commercial fermentation settings are some of the main challenges. This review provides an industry-specific summary of CRISPR–Cas9 applications in microbial fermentation and highlights technical developments, persisting challenges, and industrial advancements.

1. Introduction

Some of the most complex and varied genetic landscapes on Earth can be found in microbial genomes, which remarkably exhibit plasticity due to mechanisms like phage interactions, mobile genetic elements, and horizontal gene transfer. These mechanisms enable functional diversity and rapid adaptation in areas like metabolism, resistance, and symbiosis [1,2,3]. The discovery of novel genes and a great deal of microbial variety by high-throughput sequencing and metagenomics has highlighted the necessity of precise genome editing technologies like CRISPR–Cas systems in order to fully use microbial potential in synthetic biology, environmental remediation, and biotechnology [4,5]. The widespread use of traditional microbial genome-editing techniques, including homologous recombination, ZFNs, and TALENs, is restricted by their limited scalability, high off-target rates, and technical complexity [6]. On the other hand, CRISPR–Cas9 systems are the favored platform for applications in microbial synthetic biology, biotechnology, and fundamental research because they provide programmable, RNA-guided genome editing with enhanced specificity, usability, and efficiency [7].
The successful application of CRISPR-based genome editing in microbial systems has greatly improved genetic engineering in both model and industrial strains of Escherichia coli, Saccharomyces cerevisiae, Clostridium ljungdahlii, and Bacillus subtilis [8]. Multiplexed editing, which targets many genes simultaneously, has become crucial for improving microbial strains and re-establishing metabolic pathways [9]. These properties have made it possible to use them for everything from the synthesis of new pharmaceuticals and the clarification of complex gene regulatory networks to the generation of biofuel and improved fermentation processes [10]. Moreover, the potential for safe and effective genome engineering has also increased thanks to advanced derivatives of the CRISPR toolkit, such as base editors, prime editors, and CRISPR interference (CRISPRi), which have made it possible to precisely modify the genome, suppress transcription, and modify epigenetics without causing double-strand breaks [11,12,13].
With an emphasis on industrial fermentation, metabolic engineering, and the microbial synthesis of biofuels, biochemicals, enzymes, and medicines, this review addresses the various applications of CRISPR–Cas9 technology in microbial systems. It emphasizes how the production of high-yield strains for biotechnological innovation has been facilitated by CRISPR, which has made precise and effective genetic changes possible across a variety of microbial hosts.

2. CRISPR–Cas9 as a Genome Editing Tool in Microbes

The adaptive immunological defense of bacteria and archaea is the source of the CRISPR–Cas9 system, which introduces double-strand breaks (DSBs) at specific genomic loci to allow for precise genome editing [14] (Figure 1). The Cas9 endonuclease and single-guide RNA (sgRNA), a combination of trans-activating crRNA (tracrRNA) and CRISPR RNA (crRNA), are the two primary parts of the system. While the enzyme detects a protospacer adjacent motif (PAM), usually 5′-NGG-3′ for Streptococcus pyogenes Cas9 (SpCas9), the sgRNA uses base pairing to guide Cas9 to its DNA target [15,16]. Cas9’s conformational activation upon PAM recognition and DNA–RNA hybrid creation allows its two nuclease domains, HNH and RuvC, to cleave both DNA strands three nucleotides upstream of the PAM [17].
Cas9 is structurally made up of two lobes: one for recognition (REC) and the other for nuclease (NUC). While the RuvC and HNH domains in the NUC lobes cleave the non-target and complementary strands, respectively, the REC lobe directs interaction with the sgRNA and target DNA. Cas9-sgRNA binds to DNA via a checkpoint mechanism that guarantees cleavage only upon the accurate identification of the target sequence and PAM [18,19]. This high specificity is used for precise nucleotide substitutions, insertions, and gene knockouts. Furthermore, species-specific codon optimization and modified Cas9 variants have increased editing efficiency in microbiological systems such as Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum [7].
After a DSB is introduced, microbial cells use either homology-directed repair (HDR) or non-homologous end joining (NHEJ) to fix the break. While NHEJ directly ligates DNA ends, frequently producing frameshift mutations via indels, HDR uses a donor template to permit precise changes like gene insertion or repair [20]. Plasmid-encoded donor templates are frequently used in microbial biotechnology to take advantage of HDR, which enables the smooth integration or substitution of resistance cassettes, regulatory elements, or metabolic genes [21]. Furthermore, advancements like prime editing, inducible Cas systems, and CRISPR-associated base editors (such as dCas9 linked with deaminases) have improved editing fidelity and expanded functionality in microbial genome editing [22].

3. Applications of CRISPR–Cas9-Based Microbial Genome Editing

CRISPR–Cas9-based genome editing is essential for improving industrial microbial performance. It is a key tool for scalable, high-yield, and precision-driven microbial bioproduction because of its applications, which include the production of biofuel and biochemicals, biopharmaceuticals and enzymes, bioplastics precursor biosynthesis, and microbial strain development for food, beverage, and probiotic functionalities. The applications are discussed in detail in the following section.

3.1. CRISPR–Cas9 in Industrial Fermentation Microbes

The productivity and adaptability of many industrial fermentation microorganisms in bioproduction processes have been improved by the high-precision genome editing made possible by CRISPR–Cas9. For large-scale production applications, these systems provide dynamic control, metabolic flux optimization, pathway integration, and targeted gene alterations, which increase yields, product quality, and strain resistance (see Table 1).

3.1.1. Escherichia coli and Its Use in Platform Chemical Production

CRISPR–Cas9-based techniques have significantly improved the metabolic engineering precision and throughput of Escherichia coli, a key microbial chassis for the biosynthesis of platform chemicals [23]. By enabling scarless genome edits and multi-locus modifications, CRISPR has been used to optimize pathways for the overproduction of compounds such as succinate, lactate, malate, 1,4-butanediol, and isobutanol [24]. Specific gene deletions, such as ldhA, pta, adhE, and pflB, have redirected carbon flux towards target metabolites, while the overexpression of key enzymes, like PEP carboxylase and malate dehydrogenase, has led to succinate titers exceeding 80 g/L. Multiplex genome editing and CRISPRi tools have further enhanced strain engineering by enabling combinatorial regulation and transcriptional tuning of branch-point genes, like gltA and aceA, improving the redox balance and yields of reduced products [25,26]. Additionally, promoter and RBS engineering guided by CRISPR has enabled fine control over aromatic biosynthesis, supporting scalable production of compounds like muconic acid and vanillin. More recently, base-editing approaches have allowed for codon-level enzyme optimization without double-strand breaks, enhancing strain stability during extended fermentation [27]. Together, these advances position E. coli as a microbial factory for the efficient production of both bulk and high-value chemicals.

3.1.2. Saccharomyces Cerevisiae and Non-Conventional Yeasts

CRISPR–Cas9 has become central to the metabolic engineering of Saccharomyces cerevisiae, enabling high-precision genomic modifications to enhance the biosynthesis of ethanol, organic acids, terpenoids, and heterologous natural products [28]. Through homology-directed repair, it supports accurate gene deletions, integrations, and promoter replacements. The targeted disruption of regulators like MIG1 and RGT1 has increased carbon flux toward engineered pathways [28]. Multiplexed editing tools, such as CRISPRm and CASCADE, allow for coordinated modifications at several loci, enabling chromosomal insertion of biosynthetic clusters. For isoprenoid overproduction, Cas9-mediated overexpression of tHMG1 and integration of crtE, crtYB, and crtI into defined loci, like HO and URA3, have improved flux through the mevalonate pathway [29]. In Yarrowia lipolytica, CRISPR–Cas9 has facilitated knockouts of competing β-oxidation genes and pathway rewiring at the malonyl-CoA node for enhanced polyketide production [30]. It has also enabled stable, multi-copy integration of cellulolytic enzyme genes to expand substrate utilization in lignocellulosic biofuel processes. Cas9-based systems tailored with tRNA and ribozyme processing have further improved transformation efficiency and help to guide RNA expression [31]. These innovations collectively establish yeasts as chassis for the sustainable production of high-value and bulk fermentation products.

3.1.3. Corynebacterium, Bacillus, Clostridium Species

By overcoming the earlier obstacles associated with complex cell walls and a lack of genetic tools, CRISPR–Cas9 has enabled precise genome modifications in industrially significant Gram-positive bacteria, including solventogenic Clostridium species, Bacillus subtilis, and Corynebacterium glutamicum [32]. Scarless deletions and promoter replacements in C. glutamicum improve cofactor regeneration and reroute metabolic fluxes to maximize amino acid production [33]. Multiplexed CRISPR editing targets multiple loci simultaneously, accelerating the cycles of strain improvement. While CRISPR interference (CRISPRi) regulates sporulation and biofilm genes to enhance fermentation stability, CRISPR–Cas9 improves enzyme synthesis in B. subtilis by modifying secretion pathways and regulatory networks [34]. By integrating heterologous solventogenic pathways and site-specifically knocking out acidogenesis genes, CRISPR–Cas9 significantly boosts biofuel titers in Clostridium. Advances in transformation techniques have increased the efficiency of CRISPR editing in these sporulating and anaerobic bacteria [35]. Overall, combined CRISPR–Cas9 platforms streamline the engineering of these species into microbial factories for amino acids, enzymes, solvents, and biofuels.

3.1.4. Lactic Acid Bacteria and Acetic Acid Bacteria

The genetic engineering of lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which are essential for food fermentation and industrial uses, has been transformed by CRISPR–Cas9 editing. CRISPR–Cas9 allows for the effective elimination of genes that produce byproducts and the integration of metabolic pathways for improved lactic acid yield and flavor compound manufacturing in LAB species like Lactococcus lactis and Lactobacillus plantarum [36]. By modifying sugar transport networks and genes for acid tolerance by precision editing using homology-directed repair, fermentation can become more resilient and efficient. Reversible gene regulation is another feature of CRISPRi systems that allows for dynamic control over metabolic flux and stress-response pathways [37]. CRISPR methods tailored to these bacteria’s distinct transformation barriers and cell envelope architectures are advantageous [38]. Workflows for developing strains are further streamlined by recent advancements in plasmid curing and markerless editing [39]. In general, CRISPR–Cas9 applications in LAB and AAB increase their usefulness as biocatalysts by enhancing the stability of industrial processes and product yield and quality.
Table 1. Microbial strains engineered using CRISPR–Cas9 for enhanced industrial bioproduction.
Table 1. Microbial strains engineered using CRISPR–Cas9 for enhanced industrial bioproduction.
Serial NumberMicrobial StrainIndustrial
Product(s)		Key Genetic TargetsEnhancement AchievedReference
1Escherichia coliSuccinic acidldhA, adhE, pta, pflBRedirected flux to TCA cycle; increased succinate yield (80 g/L)[40]
2Saccharomyces cerevisiaeIsoprenoids (e.g., β-carotene)tHMG1, ERG20, crtE/I/YBIncreased Isoprenoid flux via MVA pathway[41]
3Yarrowia lipolyticaFatty acids, polyketidesβ-oxidation genes, POX, LIP familyHigh lipid & polyketide productivity[42]
4Corynebacterium glutamicumLysine, GlutamateNADPH supply genes, promoter regionsCofactor balancing; increased amino acid titers [43]
5Bacillus subtilisEnzymes, vitaminsSecretion genes (aprE, nprE)Improved extracellular protein secretion[44]
6Clostridium beijerinckiin-ButanoladhE1, adhE2 (solvent genes)Boosted solventogenesis; stable yields[45]
7Clostridium acetobutylicumAcetone-butanol-ethanol (ABE)Acid formation genes Reduced acid byproducts; increased butanol:acetate ratio[46]
8Lactococcus lactisLactic acid, aroma compoundsldh, adhE, sugar transportersOptimized flavor compound production[47]
9Gluconobacter oxydansSorbitol, acetic acidRespiratory dehydrogenasesImproved oxidative biotransformation[48]
10Synechocystis sp.IsobutanolalsS, ilvC, kivDCyanobacterial redirection of carbon flux [49]

3.2. Biofuels and Biochemicals Production

Biofuels, derived from biomass, offer a cost-saving and eco-friendly solution for depleting fossil fuel stocks. Because of their numerous advantages, businesses, policymakers, and scientists are increasingly focusing their attention upon inexhaustible and clean energy sources such as bioethanol and biodiesel [50]. In recent years, there has been growing discussion about potential replacements for transport fuels, such as petroleum, using biofuels [51]. Microorganisms, such as Clostridium thermocellum bacteria and fungi like Neurospora crassa, Fusarium oxysporum, and S. cerevisiae and Paecilomyces sp. [52] serve as potential production venues for bio-based products such as bioethanol and biobutanol products. The CRISPR–Cas9 system is one of the most recent genetic modification methods that have transformed the industry into one that enables targeted, multiplexed, and proficient genome modification [53]. The present section discusses the use of CRISPR–Cas9 in three areas that are of paramount significance: the commercial scale-up of microbial biofuel systems; increasing stress tolerance; and metabolic pathway engineering. Table 2 presents a list of the strains of microorganisms used for increased biofuel production that are CRISPR–Cas9-mediated.

3.2.1. Metabolic Pathway Engineering

The fine-tuning of microbial metabolic pathways is crucial for effective biofuel production. CRISPR–Cas9 allows for targeted editing, which can redirect metabolic flows to the desired biofuels [53]. In recent years, the CRISPR–Cas9 system engineered a dual-operon-based synthetic pathway in the genome of Escherichia coli strain MG1655, which generated 5.4 g/L n-butanol in a medium with glucose as the carbon source. This pathway was then replicated in an ethanologenic strain of E. coli strain SSK42 to convert xylose into butanol via a redox balanced pathway [54]. Following the deletion of the gene encoding endogenous ethanol synthesis, a synthetic butanol cassette was incorporated into the genome of E. coli strain SSK42 using the CRISPR–Cas9 system. A total of 4.32 g/L butanol was produced by the recently modified strain ASA02 in a batch-fed fermentation process [54].
In Clostridium saccharoperbutylacetonicum, the two key genes that encode for the synthesis of acetate and butyrate: pta and buk, were targeted by the CRISPR–Cas9 system in order to alter N1-4, a strain known to produce hyper butanol [55]. In the batch fermentation, increased yield, butanol synthesis, and mutant selectivity were achieved; however, the fermentation medium employed determined these results. At 19.0 g/liter, the P2 medium produced the maximum butanol output in the batch fermentation method [55]. The PJ23119 promoter was used to increase the efficiency of gRNA (gRNA) expression guidance.
Wasels et al. created a dual plasmid-inducible CRISPR–Cas9 genome editing method [56] for the solventogenic bacterium Clostridium acetobutylicum ATCC 824, resulting in mutants that generated an isopropanol–butanol–ethanol combination. Figure 2 summarizes the core steps involved in CRISPR–Cas9-mediated genome editing using industrial microbes.
Considering the recent advantages of the CRISPR–Cas9 technology, metabolic engineering is still in its early stages, and it necessitates an advanced degree of expertise. Advances in high-throughput technologies like transcriptomics, proteomics, and metabolomics have led to a growing understanding of the expression patterns of particular genes and their function in metabolic pathways linked to the synthesis of biofuel [57].

3.2.2. CRISPR for Tolerance Engineering

Among the most prominent barriers to microbial biofuel production is product toxicity caused by accumulation and a lack of fermentative conditions. Butanol or ethanol levels in excess of certain thresholds can become toxic to the cell membrane, inhibit the optimal function of enzymes, and, in extreme cases, even kill the host. To preempt such barriers, CRISPR–Cas9 was used to produce stress-resistant organisms [51].
Genetic strategies have been utilized to improve ethanol tolerance in Zymomonas mobilis. Of particular interest is the fact that overexpression of the RNA chaperone-coding hfq gene has been found to improve tolerance to a significant degree. This enhancement is primarily attributed to the reduced reactive oxygen species (ROS) produced under ethanol-induced stress and energy conservation in cells by suppressing flagellar growth and heat shock proteins [58]. In recent research, the use of CRISPR–Cas9 for designing stress-tolerance circuits in Saccharomyces cerevisiae has been shown to significantly improve its productivity and robustness in industrial lignocellulosic fermentation. Target genes (groES, dnaK, katG, and sod) that respond to heat shock and oxidative stress have been used to enhance cellular tolerance to challenging conditions [59]. Under harsh industrial fermentation conditions, genome editing using CRISPR–Cas9 has also been used to build the insertion of precise mutations to improve membrane integrity or decrease permeability to harmful substances, improving both microbial lifespan and biofuel titers. Through the regulation of gene silencing in a reversible fashion, without permanently altering the genome, CRISPR interference (CRISPRi) provides a convenient shortcut to gene knockouts. The regulation of such crucial stress-response genes in this fashion is particularly advantageous, as cell productivity and fitness are dependent upon the sharp regulation of repression in its totality [60,61].
Table 2. Strains of microorganisms with metabolic engineering that produce biofuel.
Table 2. Strains of microorganisms with metabolic engineering that produce biofuel.
S. NoOrganisms ProductPathwaySubstrateCRISPR Tool UsedReferences
1Corynebacterium glutamicum3-Hydroxypropionic acidGlycerol Pathway Glucose, xyloseCRISPR–Cas9[62]
2Clostridium autoethanogenumEthanolFerredoxin oxidoreductaseSynthetic mediumCRISPR–Cas12a[63]
3Synechocystis sp.IsobutanolEhrlich pathwayGlucoseCRISPR interference (CRISPRi)[64]
4S. elongates1,3-PropanediolSynthetic metabolic pathwaySynthetic mediumMultiplex CRISPR editing[65]
5E. coliFatty alcoholFatty acyl-ACP reductase-dependentSynthetic mediumCRISPR–Cas9[66]
6Saccharomyces cerevisiae2,3-ButanediolButanediol biosyntheticGlucose, galactoseCRISPRi[67]
7Klebsiella pneumoniae2-ButanolMeso-2,3-butanediol synthesisGlucoseCRISPRa[68]
8C. cellulolyticumn-ButanolCoA-dependentCelluloseCRISPR–Cas9[69]
9Thermoanaerobacterium saccharolyticumn-Butanoln-butanolXylose CRISPR–Cas12a[70]
10S. cerevisiaeIsobutanolEmbden-MeyerhofSynthetic mediumBase Editing[71]
11Clostridium Tyrobutyricumn-ButanolXylose metabolic pathwayGlucose and XyloseCRISPRi[72]
12C. cellulovoransEthanol, n-ButanolFatty acyl-ACP reductase-dependentCelluloseCRISPR–Cas9[73]

3.3. Microbial Production of Biopharmaceuticals and Enzymes

The microbial biosynthesis of industrial enzymes and biopharmaceuticals has been transformed by CRISPR–Cas9 technology, which allows for multiplex and precise genome changes, simplifying strain development and pathway optimization. The manufacture of therapeutic proteins, monoclonal antibodies, vaccines, hormones, and industrial enzymes relies heavily on microbial platforms, especially bacteria and yeast, because of their scalable fermentation processes, well-characterized genomes, and fast growth rates [74].

3.3.1. Recombinant Therapeutic Proteins in Bacteria and Yeast

One important class of biopharmaceuticals produced through microbial fermentation consists of recombinant therapeutic proteins such as hormones, cytokines, antibodies, and vaccines [75]. The well-characterized genomes, ease of genetic manipulation, and cost-effective scalability of bacteria and yeast make them widely used hosts. CRISPR–Cas9-based genome editing has enabled unprecedented precision and efficiency in engineering these organisms for optimal recombinant protein production [8]. In bacterial systems, CRISPR–Cas9 has been used to integrate transgenes directly into chromosomal loci with strong, constitutive promoters, eliminating the need for plasmids and the associated instability and metabolic burden. Integration into transcriptionally active regions ensures high expression levels, while concurrent deletion of proteases (e.g., ompT, lon) enhances protein stability [76]. CRISPR editing can also be combined with codon optimization and signal peptide engineering to improve translation and secretion [77].
Yeast systems, particularly Saccharomyces cerevisiae and Pichia pastoris, are well-suited for producing complex proteins requiring post-translational modifications [78,79]. CRISPR–Cas9 enables the precise integration of multigene constructs and facilitates human-like glycosylation by replacing native glycosylation pathways. It also allows for fine-tuning of the unfolded protein response (UPR) through the modification of genes, such as HAC1 and KAR2, thereby enhancing protein folding and secretion efficiency [80]. Current approaches combine metabolic modeling with CRISPR interference/activation (CRISPRi/a) to dynamically regulate gene expression, improving precursor availability, energy balance, and protein folding capacity [81]. This multiplexed, systems-level engineering significantly accelerates the development of efficient microbial strains for therapeutic protein production [9].

3.3.2. Industrial Enzyme Engineering and Optimization

CRISPR-mediated genome engineering has revolutionized the microbial production of industrial enzymes. Rational design strategies enabled by CRISPR–Cas tools are gradually replacing traditional trial-and-error mutagenesis approaches [82]. These technologies have significantly enhanced the production of enzymes, including cellulases for biofuels; proteases for detergents; and amylases for food processing. CRISPR enables precise and targeted improvements in host cell function and enzyme expression, in contrast to random mutagenesis [83]. For example, in Bacillus subtilis, CRISPR was used to substitute native promoters with stronger synthetic ones, resulting in increased α-amylase production without overburdening the host’s metabolic capacity [84]. Similarly, in Escherichia coli, deletion of transcriptional repressors redirected carbon flux, boosting the synthesis of thermostable lipases. These targeted interventions improve pathway efficiency and prevent the buildup of toxic intermediates [85].
Additionally, CRISPR enhances enzyme secretion and stability. In Pichia pastoris, the disruption of vacuolar protease genes (PEP4, PRB1) elevated extracellular phytase levels [86]. In E. coli, the co-expression of molecular chaperones, such as groEL and dnaK, alleviates protein folding stress during protease production [87]. These helper genes can be integrated into the host genome using CRISPR, without the need for antibiotic resistance markers. Genome-wide CRISPR knockout and CRISPRi libraries also facilitate the identification of key genes influencing redox balance, energy metabolism, and stress responses [88]. For instance, in Trichoderma reesei, CRISPR was used to modify regulators involved in glucose repression, leading to enhanced cellulase production under industrial fermentation conditions [89].
Recent advances, such as CRISPR base editors, allow for single-nucleotide modifications to enzyme-coding genes. For example, site-directed mutations in xylanase improved thermal stability without compromising catalytic activity. These precise modifications streamline enzyme optimization for specific industrial applications [90].

3.3.3. CRISPR for Pathway Balancing and Yield Improvement

The efficient microbial production of target compounds requires balanced metabolic pathways. CRISPR–Cas systems offer dynamic and modular control over gene expression, enabling the rerouting of metabolic flux and the removal of bottlenecks across biosynthetic networks [91]. One approach involves CRISPR activation (CRISPRa) and interference (CRISPRi), which modulate gene expression without altering DNA sequences [92]. For instance, in Corynebacterium glutamicum, CRISPRi downregulated pyruvate dehydrogenase, redirecting carbon flux toward L-lysine biosynthesis and boosting production by over 30% [93]. In Yarrowia lipolytica, CRISPRa was employed to enhance the expression of NADPH-generating enzymes, thereby improving fatty acid synthesis [94]. Multiplex CRISPR arrays enable the simultaneous editing or regulation of multiple genes. In E. coli, this approach targeted both ATP-consuming and competing metabolic pathways, leading to increased yields of succinate and polyhydroxybutyrate (PHB) [95]. These combinatorial strategies help to maintain metabolic balance and avoid the accumulation of toxic intermediates that can limit product titers.
To ensure strain stability during large-scale fermentation, CRISPR has been applied to delete the insertion sequences, prophages, and mobile genetic elements that are common sources of genomic instability. For example, in Clostridium acetobutylicum, such deletions improved the consistency of solvent production during extended fermentation runs [96]. The glycoengineering techniques applied to plant cells to humanize their N-glycan structures are shown schematically in Figure 3. Advanced methods, like combinatorial pathway optimization (CPO) integrate CRISPR tools with machine learning and guide RNA libraries to predict optimal gene targets for overexpression or knockout [97]. This approach was used to fine-tune the mevalonate pathway in Saccharomyces cerevisiae, significantly increasing the production of isoprenoids, such as artemisinic acid, a key precursor for antimalarial drugs [98].

3.4. CRISPR-Powered Advances in the Fermentation of Food

One of the key applications of CRISPR–Cas9 is food and agricultural science. The food sector has benefited significantly from modern technological advances in genetic engineering over the past decades. Modern genetic technologies have enhanced both agronomic and non-agronomic attributes, including the nutritional and sensory value of crops and agronomically significant attributes such as herbicide tolerance, insect resistance, grain content, plant height and weight. Due to all these advances, food products with enhanced attributes are produced at a larger scale [99]. CRISPR supplies advanced tools to improve the attributes of microorganisms, manage the metabolic processes, and produce second-generation functional foods used in the dairy, brewery, and probiotic food sectors. The CRISPR–Cas9 system enables precise genome editing in industrial microbes. As illustrated in Figure 4, gene insertion and deletion can be efficiently achieved through the electroporation of plasmids encoding Cas9, guide RNAs, and donor DNA This section illustrates a few significant applications of CRISPR–Cas9 in food fermentation such as the production of probiotic microbes and the upgrading of starter cultures.

3.4.1. Improvement in Dairy and Brewing Starters

Starter cultures are used in the manufacturing process of fermented food, such as yogurt and cheese, and beverages such as wine and beer. CRISPR–Cas9 enables targeted gene insertions and deletions, regulation over microbial genomes, and the optimization of metabolism and resistance functions. Lactic acid bacteria (LAB), such as Lactococcus lactis and Lactobacillus species are susceptible to attack from the phage, which can lead to the spoilage of fermentation batches. CRISPR-based phage-resistance cassettes have emerged as a solution to defend the starter strains and obtain fermentation stability [100]. Saccharomyces cerevisiae has been genetically improved using CRISPR to boost ethanol tolerance, reduce off-flavors, such as diacetyl and other undesired compounds, and manage the metabolism of carbohydrates. Gene editing to target maltose and maltotriose transport enhanced the fermentation efficiency and the production of ethanol [101].

3.4.2. CRISPR in Probiotic and Functional Microorganism Engineering

The international probiotic and functional food market is expanding rapidly with the direction of health-conscious consumers. CRISPR enabled the targeted design of probiotic strains, such as Lactobacillus and Bifidobacterium, to maximize stress resistance, the potential to colonize, and bioactive compound production [102]. CRISPR, for instance, has been used to enhance acid and bile tolerance in Lactobacillus plantarum so that it can survive through the gut. Folate, vitamin B12, or GABA production genes have been overexpressed to design strains with additional health benefits [103,104]. CRISPR-interference (CRISPRi) has also been used to fine-tune gene expression while maintaining the permanent installation of mutations to maintain GRAS (generally recognized as safe) status. These technologies pave the way to the next era of functional foods, providing sustenance but actually bringing about health [105].
CRISPR–Cas9 is transforming the fermentation of food and beverages through the potential to design microbials rapidly and specifically, but safely. From increasing starter culture robustness and flavor profiles to the development of improved probiotics, this gene editing method is laying the groundwork for a more sustainable and healthy food and beverage sector. Rapid developments in CRISPR tools, such as the use of base editing and prime editing, can, in the near future, further tailor microbial functions with increased precision.

4. Computational Tools and Predictive Modeling for CRISPR Design in Microorganisms

The advent of CRISPR–Cas technology has transformed gene editing with unprecedented effectiveness and precision [106]. An essential building block for successful gene editing in microbial biotechnology is creating potent single-guide RNAs (sgRNAs). Towards this end, numerous computational methods and prediction models were established for optimizing sgRNA for maximum on-target performance and minimal off-target activities [107].

4.1. Improved sgRNA Design Tools

Recent times have seen the development of high-performance computational platforms specifically for microbial sgRNA design. A prime example is CRISPRedict, which offers interpretable models for predicting sgRNA efficacy with accuracy and intuitive interfaces [108]. Likewise, GLiDe enables genome-scale CRISPR interference (CRISPRi) sgRNA design for prokaryote genomes, enabling large-scale functional genomics experiments [109].

4.2. Integrating Deep Learning and Artificial Intelligence

There has been a remarkable improvement in predictive accuracy by incorporating deep learning into CRISPR toolkit development. DeepFM-Crispr, for instance, utilizes transformer-based models for forecasting the on-target effectiveness and off-target implications for Cas13d platforms [110]. A case in point is CRISPR-GPT, which represents large language model integration into field knowledge for automating gene-editing experiment planning as well as advising scientists on selecting pertinent CRISPR platforms, the prediction of sgRNA, and the development of confirmation procedures [111].

4.3. Challenges and Future Directions

Despite all these advances, challenges persist. Off-target effects continue to present a problem, requiring the ongoing optimization of prediction models. Added to this, inconsistencies between microbial species reaffirm that species-specific models will be required. Efforts in the future should be oriented toward integrating multi-omics for enhanced accuracy predictions and toward building tools that can cope with novel emerging genomic contexts across various microorganisms [107,108].
Ultimately, predictive modeling and computational aids together have tremendously eased CRISPR design for microbes. With further advances in the future, these aids will increasingly be involved in microbial synthetic biology and microbial biotechnology.

5. Challenges, Limitations, and Ethical Considerations

5.1. Off-Target Effects

Although Cas9 cuts DNA at certain targets based on the 20-letter guide RNA (gRNA) sequence and the nearby pam sequence, it may sometimes make cuts in incorrect places in the genome. This occurs when the gRNA unintentionally sticks to an identical-looking DNA sequence somewhere else in the chromosome. These off-target cuts can cause sudden DNA changes, which makes using CRISPR more challenging in different organisms [112].

5.2. Efficiently Editing Microbial Genome

Modifying genes using CRISPR is challenging, mainly in complex environments with many dissimilar microbes. Traditional methods of delivering CRISPR tools may not reach all the target cells, leading to low editing accomplishment. Sometimes CRISPR also changes parts of DNA that it should not, which reduces the accuracy and safety of a process [113]. Unintended changes in genomic areas that are not the intended targets, referred to as off-target effects, present a major obstacle, as these effects can undermine the precision and safety of genome editing techniques [114].
Microbial genomes can be quite complex, as they often have repeated DNA parts, mostly of G and C bases with special structures. These characteristics can make it difficult to precisely find and edit specific spots in their DNA [115]. Over time, microbes become resistant to CRISPR-based methods, which makes gene editing less effective. There are also ethical concerns, most importantly when microbes are used in industry or in the environment. This can be made effective by using better methods such as advance vectors or nanoparticles [116].

5.3. Host Immune Responses

Initially adaptive immune responses to Cas9 proteins present a crucial obstacle for CRISPR-based therapies, as many individuals possess antibodies and T cells targeting Staphylococcus aureus and Streptococcus pyogenes Cas9 variants. These immune responses can make gene editing less effective and increase the chance of side effects that are caused by the immune system. To control these challenges, approaches like utilizing immune-orthogonal Cas9 variants, modifying proteins to lower immunogenicity, and applying transient immunosuppressive treatments have been investigated [117].

5.4. Delivery Challenges in CRISPR–Cas9 Gene Therapy

The fundamental barrier to using CRISPR–Cas9 for in vivo gene therapy is making sure of a delivery method that is both biocompatible and secure. The compelling issue is the susceptibility of CRISPR–Cas9 components to degradation by nucleases and their quick clearance by immune cells like macrophages. The large size of the Cas9 protein, its low positive charge, and the water-soluble nature of single-guide RNA (sgRNA) pose substantial exceptions for effective nuclear entry and gene transfer [77]. The most common motion involves directly administering the Cas9 protein alongside sgRNA; this method is complicated to apply in vivo as the large size of Cas9 increases the risk of triggering an immune response [118].

6. Future Directions

6.1. Integration with AI, Automation, and HTS

The potential for future microbial technology applications including CRISPR lies in its integration into high-throughput screening (HTS) technology, laboratory automation, and artificial intelligence (AI). Guide RNA (gRNA) predictions, off-target effect avoidance, and metabolic pathway designs are optimized in silico through machine learning models prior to laboratory experiments [119]. Automated processes for CRISPR cloning, transformation, and robot-based screening have significantly reduced strain cycle times. Additionally, HTS platforms enable fast screening for tens of thousands of engineered variants for traits such as product yield, tolerance, or growth rates. Such combined approaches reduce experimental cost, minimize the time-to-market, and allow for iterative strain cycle optimization for industrial fermentation.

6.2. CRISPR 3.0 Tools for Microbial Cell Factory Design

The toolkit for CRISPR is shifting from mere simple genome editing (CRISPR 1.0) and gene regulation (CRISPR 2.0) toward multiplexed, programmable, and reversible modification of genomes (CRISPR 3.0). To date, base editors, prime editors, and CRISPRa/i (activation or interference) are available for tuning genes with high accuracy, without inducing double-stranded DNA breaks [120]. Such platforms, within microbial cell factories, can be applied for tuning complex metabolism networks, inserting combinatorial libraries, and dynamic modulation through synthetic pathways. For example, multiplex base editing in the case of E. coli and S. cerevisiae has enhanced strains for tolerating solvents and amino acid and alcohol production [97].

6.3. Next-Generation Fermentation System Vision

The next-generation fermentation platforms will include intelligent, adaptive, and auto-optimal microbial platforms. Engineered microbes will be endowed with biosensors based on CRISPR and feedback mechanisms, enabling the real-time measurement of metabolic load; the availability of nutrients or toxicants; and gene expression adjustment. With AI–control system integrated bioreactors, we will introduce an era for autonomous fermentation [102]. The facility for creating synthetic consortia with more than one strain working interdependently through CRISPR-mediated networks will allow for a broader diversity of feedstocks as well as products. These developments will be key in initiating a paradigm shift toward petrochemical-free manufacturing and carbon-neutral biomanufacturing.

7. Conclusions

CRISPR–Cas9 has shown itself to be an innovative technology in microbial genome engineering and is proving to be a highly accurate, versatile, and systematic method for genetic modification. This review has highlighted its transformative role in microbial fermentation, allowing for the development of engineered strains that crucially boost the production of biofuels, pharmaceuticals, and industrial enzymes. The technology proceeds to advance through the acceptance of novel techniques, like base editing, prime editing, and CRISPRi/a, engaging its utility through diverse microbial systems. Future development points to a combination of CRISPR–Cas9 with artificial intelligence (AI) and machine learning usable models, such as deepHF, TIGER, and CRISPRon, to amplify the precision of guide RNA selection, enhancing both target accuracy and decreasing unintended edits. The advance of CRISPR 3.0 tools and intelligent fermentation systems enable adaptive gene regulation and real-time regulation over metabolic flux. Beyond fermentation, CRISPR technology has broader applications in sectors including agriculture, through trait improvements in crops; in oncology, by exposing therapeutic targets and resistance mechanism; and in obtainable energy through metabolic pathway engineering in microalgae for biofuel production. Although it has broad potential, CRISPR–Cas9 faces evolving challenges, including off-target effects, delivery limitations, and the development of a regulatory framework. Continuous research and innovation are fast overcoming these barriers, steering the technology towards preservation and more effective deployment.
In summary, CRISPR–Cas9 is a foundational element in the fields of synthetic and industrial biotechnology. Its future, when reconciled with AI, system biology, and ecological design principles, holds great promise for delivering adaptable, sustainable and impressive solutions to global problems in medicine, agriculture, and environmental management.

Author Contributions

C.D.: Original draft preparation, writing, reviewing and editing; A.M.: Writing, reviewing and editing; A.A.: Writing, reviewing and editing; A.K.J.: Conceptualization, writing, reviewing and editing; P.P.S.: Conceptualization, writing, reviewing and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Mechanism of the CRISPR–Cas9 adaptive immune system in microbial genome.
Figure 1. Mechanism of the CRISPR–Cas9 adaptive immune system in microbial genome.
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Figure 2. Schematic overview of the CRISPR–Cas9 genome editing workflow in industrial microbes.
Figure 2. Schematic overview of the CRISPR–Cas9 genome editing workflow in industrial microbes.
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Figure 3. CRISPR–Cas9 enables the precise engineering of microbes for therapeutic proteins.
Figure 3. CRISPR–Cas9 enables the precise engineering of microbes for therapeutic proteins.
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Figure 4. Gene deletion via RecT-assisted CRISPR–Cas9 in Lactococcus lactis.
Figure 4. Gene deletion via RecT-assisted CRISPR–Cas9 in Lactococcus lactis.
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Dudeja, C.; Mishra, A.; Ali, A.; Singh, P.P.; Jaiswal, A.K. Microbial Genome Editing with CRISPR–Cas9: Recent Advances and Emerging Applications Across Sectors. Fermentation 2025, 11, 410. https://doi.org/10.3390/fermentation11070410

AMA Style

Dudeja C, Mishra A, Ali A, Singh PP, Jaiswal AK. Microbial Genome Editing with CRISPR–Cas9: Recent Advances and Emerging Applications Across Sectors. Fermentation. 2025; 11(7):410. https://doi.org/10.3390/fermentation11070410

Chicago/Turabian Style

Dudeja, Chhavi, Amish Mishra, Ansha Ali, Prem Pratap Singh, and Atul Kumar Jaiswal. 2025. "Microbial Genome Editing with CRISPR–Cas9: Recent Advances and Emerging Applications Across Sectors" Fermentation 11, no. 7: 410. https://doi.org/10.3390/fermentation11070410

APA Style

Dudeja, C., Mishra, A., Ali, A., Singh, P. P., & Jaiswal, A. K. (2025). Microbial Genome Editing with CRISPR–Cas9: Recent Advances and Emerging Applications Across Sectors. Fermentation, 11(7), 410. https://doi.org/10.3390/fermentation11070410

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