Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable “Clean” Mutant Selection in <i>Clostridium beijerinckii</i> as an Example

Wang, Yi; Zhang, Zhong Tian; Seo, Seung Beom; Patrick, Lynn; Lu, Ting; Liu, Jing Jing; Blaschek, Hans P.

doi:10.1021/acssynbio.6b00060

Cited by 154 publications

(162 citation statements)

References 38 publications

(94 reference statements)

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“…So far, the CRISPR-Cas9 system has been applied in very few bacterial species to edit their genomes (9)(10)(11)(12)(13)(14)(15)). An example is the two-plasmid-based system constructed for engineering Escherichia coli (10).…”

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confidence: 99%

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Altenbuchner

2016

Appl Environ Microbiol

271

246

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The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temperature-sensitive replication origin of plasmid pE194 ts for B. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries the cas9 gene under the control of the B. subtilis mannose-inducible promoter P manP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5= end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B. subtilis chromosome and by repair of the trpC2 mutation of B. subtilis 168. IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing of B. subtilis overcomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

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confidence: 99%

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Altenbuchner

2016

Appl Environ Microbiol

271

246

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“…Then, through employing a second sgRNA that recognizes the introduced artificial protospacer sequence, the target sites will be mutated, and meanwhile the artificial PAM or protospacer sequence will be back-mutated to its original sequence, generating a clean mutant with only the target sites mutated (Biot-Pelletier and Martin, 2016). Wang et al (2016) reported a similar approach. The PAM or protospacer used in the first step was modified or deleted, whereas the artificial PAM was created at the target site.…”

Section: Resultsmentioning

confidence: 90%

A Novel and Efficient Method for Bacteria Genome Editing Employing both CRISPR/Cas9 and an Antibiotic Resistance Cassette

et al. 2017

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As Cas9-mediated cleavage requires both protospacer and protospacer adjacent motif (PAM) sequences, it is impossible to employ the CRISPR/Cas9 system to directly edit genomic sites without available PAM sequences nearby. Here, we optimized the CRISPR/Cas9 system and developed an innovative two-step strategy for efficient genome editing of any sites, which did not rely on the availability of PAM sequences. An antibiotic resistance cassette was employed as both a positive and a negative selection marker. By integrating the optimized two-plasmid CRISPR/Cas system and donor DNA, we achieved gene insertion and point mutation with high efficiency in Escherichia coli, and importantly, obtained clean mutants with no other unwanted mutations. Moreover, genome editing of essential genes was successfully achieved using this approach with a few modifications. Therefore, our newly developed method is PAM-independent and can be used to edit any genomic loci, and we hope this method can also be used for efficient genome editing in other organisms.

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“…Recent advances in the genetic manipulation of clostridia (primarily C. acetobutylicum ATCC 824 and C. beijerinckii NCIMB 8052) have afforded several methods for targeted gene disruption, including (i) heterologous expression of mobile group II introns with the ClosTron system (36), (ii) allelic exchange via homologous recombination (37,38), and very recently, (iii) clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas9 genome editing (39)(40)(41). Given the observed instability and off-target effects associated with mobile group II introns (42) as well as the start-up time required to adapt an efficient CRISPR-Cas9 system for use in C. saccharoperbutylacetonicum N1-4, we chose here to pursue a double-crossover-based allelic exchange (DCAE) method.…”

Section: Resultsmentioning

confidence: 99%

“…This study serves as a foundation for future metabolic engineering efforts of this prominent solvent producer to not only improve fermentation performance metrics (e.g., solvent titers, productivities, and yields, oxygen tolerance, substrate specificity, cell viability), but also to serve as a platform for the production of other high-value products (e.g., higher alcohols and petrochemical precursors). Additionally, this study opens the door for advanced genetic manipulations of this organism using recent achievements in adapting CRISPR/Cas9 systems for use in clostridia (40,41). Before this is possible, future studies are required to identify additional promoters, antibiotic markers, and counterselection methods able to function in C. saccharoperbutylacetonicum N1-4.…”

Section: Discussionmentioning

confidence: 99%

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

Herman

Bedi

et al. 2017

Appl Environ Microbiol

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While a majority of academic studies concerning acetone, butanol, and ethanol (ABE) production by Clostridium have focused on Clostridium acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyperproducer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021. Following initial failures, we found that the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I), which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency, 1.1 ϫ 10 6 CFU/g DNA), we performed targeted gene deletions in C. saccharoperbutylacetonicum N1-4 using a homologous recombinationmediated allelic exchange method. Using plasmid-based gene overexpression and targeted knockouts of key genes in the native acetone-butanol-ethanol (ABE) metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case an engineered strain (Clostridium saccharoperbutylacetonicum strain N1-4/pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (encoded by adh11 and adh5) displayed 8.5-and 11.8-fold increases (respectively) in batch ethanol production.IMPORTANCE This paper presents the first steps toward advanced genetic engineering of the industrial butanol producer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT). In addition to providing an efficient method for introducing foreign DNA into this species, we demonstrate successful rational engineering for increasing solvent production. Examples of future applications of this work include metabolic engineering for improving desirable industrial traits of this species and heterologous gene expression for expanding the end product profile to include high-value fuels and chemicals.KEYWORDS ABE fermentation, Clostridium, biofuel, genetics

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Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable “Clean” Mutant Selection in Clostridium beijerinckii as an Example

Cited by 154 publications

References 38 publications

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

A Novel and Efficient Method for Bacteria Genome Editing Employing both CRISPR/Cas9 and an Antibiotic Resistance Cassette

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

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