Development of an Efficient Genome Editing Tool in
                    Bacillus licheniformis
                    Using CRISPR-Cas9 Nickase

ABSTRACT Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN , which codes for nattokinase in Bacillus subtilis , was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-S sacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-S sacC . Finally, the engineered strain DWc9nΔ7 (Δ epr Δ wprA Δ mpr Δ aprE Δ vpr Δ bprA Δ bacABC ), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis . This tool could be applied to strain improvement for future research. IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in Bacillus licheniformis via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.

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CRISPR-Cpf1-Assisted Multiplex Genome Editing and Transcriptional Repression in Streptomyces

Applied and Environmental Microbiology ◽

10.1128/aem.00827-18 ◽

2018 ◽

Vol 84 (18) ◽

Cited By ~ 35

Author(s):

Lei Li ◽

Keke Wei ◽

Guosong Zheng ◽

Xiaocao Liu ◽

Shaoxin Chen ◽

...

Keyword(s):

Natural Products ◽

Genetic Engineering ◽

Genome Editing ◽

Transcriptional Repression ◽

High Efficiency ◽

Streptomyces Coelicolor ◽

Genome Mining ◽

Strain Improvement ◽

Strand Break ◽

Content Type

ABSTRACT Streptomyces has a strong capability for producing a large number of bioactive natural products and remains invaluable as a source for the discovery of novel drug leads. Although the Streptococcus pyogenes CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in Streptomyces, it has a number of limitations, including the toxicity of SpCas9 expression in some important industrial Streptomyces strains and the need for complex expression constructs when targeting multiple genomic loci. To address these problems, in this study, we developed a high-efficiency CRISPR-Cpf1 system (from Francisella novicida) for multiplex genome editing and transcriptional repression in Streptomyces. Using an all-in-one editing plasmid with homology-directed repair (HDR), our CRISPR-Cpf1 system precisely deletes single or double genes at efficiencies of 75 to 95% in Streptomyces coelicolor. When no templates for HDR are present, random-sized DNA deletions are achieved by FnCpf1-induced double-strand break (DSB) repair by a reconstituted nonhomologous end joining (NHEJ) pathway. Furthermore, a DNase-deactivated Cpf1 (ddCpf1)-based integrative CRISPRi system is developed for robust, multiplex gene repression using a single customized crRNA array. Finally, we demonstrate that FnCpf1 and SpCas9 exhibit different suitability in tested industrial Streptomyces species and show that FnCpf1 can efficiently promote HDR-mediated gene deletion in the 5-oxomilbemycin-producing strain Streptomyces hygroscopicus SIPI-KF, in which SpCas9 does not work well. Collectively, FnCpf1 is a powerful and indispensable addition to the Streptomyces CRISPR toolbox. IMPORTANCE Rapid, efficient genetic engineering of Streptomyces strains is critical for genome mining of novel natural products (NPs) as well as strain improvement. Here, a novel and high-efficiency Streptomyces genome editing tool is established based on the FnCRISPR-Cpf1 system, which is an attractive and powerful alternative to the S. pyogenes CRISPR-Cas9 system due to its unique features. When combined with HDR or NHEJ, FnCpf1 enables the creation of gene(s) deletion with high efficiency. Furthermore, a ddCpf1-based integrative CRISPRi platform is established for simple, multiplex transcriptional repression. Of importance, FnCpf1-based genome editing proves to be a highly efficient tool for genetic modification of some important industrial Streptomyces strains (e.g., S. hygroscopicus SIPI-KF) that cannot utilize the SpCRISPR-Cas9 system. We expect the CRISPR-Cpf1-assisted genome editing tool to accelerate discovery and development of pharmaceutically active NPs in Streptomyces as well as other actinomycetes.

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Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus

Microbial Genomics ◽

10.1099/mgen.0.000669 ◽

2021 ◽

Vol 7 (11) ◽

Cited By ~ 1

Author(s):

Juan Pablo Gomez-Escribano ◽

Lis Algora Gallardo ◽

Kenan A. J. Bozhüyük ◽

Steven G. Kendrew ◽

Benjamin D. Huckle ◽

...

Keyword(s):

Genome Editing ◽

Type Strain ◽

High Efficiency ◽

Genetic Manipulation ◽

Sequence Data ◽

Streptomyces Clavuligerus ◽

Sequence Coverage ◽

Content Type ◽

Large Deletions ◽

Low Levels

Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid’s parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

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CRISPR-Cas9D10A Nickase-Assisted Genome Editing in Lactobacillus casei

Applied and Environmental Microbiology ◽

10.1128/aem.01259-17 ◽

2017 ◽

Vol 83 (22) ◽

Cited By ~ 54

Author(s):

Xin Song ◽

He Huang ◽

Zhiqiang Xiong ◽

Lianzhong Ai ◽

Sheng Yang

Keyword(s):

Genome Editing ◽

Lactobacillus Casei ◽

Genome Engineering ◽

Fluorescent Protein ◽

Genetic Manipulation ◽

Gene Knockout ◽

Single Gene ◽

Egfp Expression ◽

Broad Host Range ◽

Content Type

ABSTRACT Lactobacillus casei has drawn increasing attention as a health-promoting probiotic, while effective genetic manipulation tools are often not available, e.g., the single-gene knockout in L. casei still depends on the classic homologous recombination-dependent double-crossover strategy, which is quite labor-intensive and time-consuming. In the present study, a rapid and precise genome editing plasmid, pLCNICK, was established for L. casei genome engineering based on CRISPR-Cas9D10A. In addition to the P23-Cas9D10A and Pldh-sgRNA (single guide RNA) expression cassettes, pLCNICK includes the homologous arms of the target gene as repair templates. The ability and efficiency of chromosomal engineering using pLCNICK were evaluated by in-frame deletions of four independent genes and chromosomal insertion of an enhanced green fluorescent protein (eGFP) expression cassette at the LC2W_1628 locus. The efficiencies associated with in-frame deletions and chromosomal insertion is 25 to 62%. pLCNICK has been proved to be an effective, rapid, and precise tool for genome editing in L. casei, and its potential application in other lactic acid bacteria (LAB) is also discussed in this study. IMPORTANCE The lack of efficient genetic tools has limited the investigation and biotechnological application of many LAB. The CRISPR-Cas9D10A nickase-based genome editing in Lactobacillus casei, an important food industrial microorganism, was demonstrated in this study. This genetic tool allows efficient single-gene deletion and insertion to be accomplished by one-step transformation, and the cycle time is reduced to 9 days. It facilitates a rapid and precise chromosomal manipulation in L. casei and overcomes some limitations of previous methods. This editing system can serve as a basic technological platform and offers the possibility to start a comprehensive investigation on L. casei. As a broad-host-range plasmid, pLCNICK has the potential to be adapted to other Lactobacillus species for genome editing.

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Challenges and Advances in Genome Editing Technologies in Streptomyces

Biomolecules ◽

10.3390/biom10050734 ◽

2020 ◽

Vol 10 (5) ◽

pp. 734 ◽

Cited By ~ 2

Author(s):

Yawei Zhao ◽

Guoquan Li ◽

Yunliang Chen ◽

Yinhua Lu

Keyword(s):

Natural Product ◽

Genome Editing ◽

Genetic Manipulation ◽

Chemical Compounds ◽

Gene Clusters ◽

Future Research ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Research Focus ◽

Experimental Conditions

The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.

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Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

mSphere ◽

10.1128/msphere.00130-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 68

Author(s):

Kyunghun Min ◽

Yuichi Ichikawa ◽

Carol A. Woolford ◽

Aaron P. Mitchell

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Genetic Analysis ◽

Genome Editing ◽

Drug Targets ◽

Gene Deletion ◽

Genetic Manipulation ◽

Content Type ◽

Biological Features ◽

Link Type

ABSTRACT The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

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Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

mBio ◽

10.1128/mbio.00308-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 69

Author(s):

Ian R. Monk ◽

Jai J. Tree ◽

Benjamin P. Howden ◽

Timothy P. Stinear ◽

Timothy J. Foster

Keyword(s):

Staphylococcus Aureus ◽

Plasmid Dna ◽

High Efficiency ◽

Recombinant Dna ◽

Genetic Manipulation ◽

Bacterial Pathogen ◽

Type I ◽

Content Type ◽

E Coli ◽

Adenine Methylation

ABSTRACTStaphylococcus aureusis a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates ofS. aureus. Here, we describe the construction and characterization of the IMXXB series ofEscherichia colistrains that mimic the type I adenine methylation profiles ofS. aureusclonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of majorS. aureuslineages.IMPORTANCEThe genetic manipulation of clinicalS. aureusisolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset ofS. aureusstrains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite ofE. colistrains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer intoS. aureus. The IMXXB series ofE. colistrains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation ofS. aureus.

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Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

Microbial Cell Factories ◽

10.1186/s12934-019-1219-5 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 10

Author(s):

Wei Shen ◽

Jun Zhang ◽

Binan Geng ◽

Mengyue Qiu ◽

Mimi Hu ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Zymomonas Mobilis ◽

Genome Engineering ◽

Gene Deletion ◽

Genetic Manipulation ◽

Strain Improvement ◽

Genomic Research ◽

Francisella Novicida ◽

A Genome

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

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Efficient Genome Editing in Bacillus licheniformis Mediated by a Conditional CRISPR/Cas9 System

Microorganisms ◽

10.3390/microorganisms8050754 ◽

2020 ◽

Vol 8 (5) ◽

pp. 754

Author(s):

Youran Li ◽

Hanrong Wang ◽

Liang Zhang ◽

Zhongyang Ding ◽

Sha Xu ◽

...

Keyword(s):

Success Rate ◽

Genome Editing ◽

Bacillus Licheniformis ◽

Genetic Manipulation ◽

Inducible Promoter ◽

Bacterial Cells ◽

Protospacer Adjacent Motif ◽

Constitutive Overexpression ◽

Short Palindromic Repeat ◽

Overexpression System

Bacillus licheniformis is widely used to produce multiple enzymes and chemicals in industrial fermentation. It is also an organism that is hard to genetically manipulate, which is mainly attributed to its extremely low transformation efficiency. The lack of genetic modification technology severely limits its further application. In this study, an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid was developed for B. licheniformis with the cas9 gene under the control of a xylose-inducible promoter. By means of this design, the expression of the cas9 gene could be repressed without xylose, which significantly improved the transformation ratio from less than 0.1 cfu/μg to 2.42 cfu/μg DNA. Compared with this conditional system, a constitutive overexpression system led to significant growth retardation in bacterial cells. Both the biomass and specific growth rate decreased greatly. After transformation, successful genome editing could be triggered by 0.5% xylose. When the α-amylase gene amyL was used as a genomic target, the efficiencies of its disruption using three different protospacer-adjacent motif (PAM) sequences were 64.3%, 70.9%, and 47.1%, respectively. Moreover, temperature plays a pivotal role in the function of the constructed CRISPR system. The maximum success rate reached 97% at 20 °C, while higher temperatures negatively impacted the function of the system. These results suggested that the design with a cas9 gene under the strict control of a xylose-inducible promoter significantly improved the success rate of genome editing in this host. This work contributes to the development of genetic manipulation and furthers the use of B. licheniformis as an efficient industrial workhorse.

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Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

mBio ◽

10.1128/mbio.00277-11 ◽

2012 ◽

Vol 3 (2) ◽

Cited By ~ 242

Author(s):

Ian R. Monk ◽

Ishita M. Shah ◽

Min Xu ◽

Man-Wah Tan ◽

Timothy J. Foster

Keyword(s):

Staphylococcus Aureus ◽

Staphylococcus Epidermidis ◽

High Efficiency ◽

Genetic Manipulation ◽

Laboratory Strain ◽

Small Subset ◽

Type I ◽

Major Barrier ◽

Content Type ◽

Functional Genomic Analysis

ABSTRACTThe strong restriction barrier present inStaphylococcus aureusandStaphylococcus epidermidishas limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain ofS. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiencyEscherichia colicloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates ofS. aureusandS. epidermidis. We also show that the loss of restriction (both type I and IV) in anS. aureusUSA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens.IMPORTANCEStaphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding ofStaphylococcus aureusandStaphylococcus epidermidisinfections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.

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A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792

Applied and Environmental Microbiology ◽

10.1128/aem.00408-20 ◽

2020 ◽

Vol 86 (13) ◽

Cited By ~ 1

Author(s):

François Wasels ◽

Gwladys Chartier ◽

Rémi Hocq ◽

Nicolas Lopes Ferreira

Keyword(s):

Genome Editing ◽

Clostridium Acetobutylicum ◽

Single Gene ◽

Bacterial Species ◽

Abe Fermentation ◽

Complex Activity ◽

Triple Mutant ◽

Alcohol Dehydrogenases ◽

Content Type ◽

Butanol Dehydrogenase

ABSTRACT Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4. Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain. IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.

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