A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

Seys, François M.; Rowe, Peter; Bolt, Edward L.; Humphreys, Christopher M.; Minton, Nigel P.

doi:10.3390/genes11040458

Cited by 13 publications

(20 citation statements)

References 21 publications

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“…The study consisted of four parts: (1) sporulation mutants of C. botulinum Group II type E strain Beluga were constructed using a novel CRISPR-Cas9 bookmark approach ( Seys et al, 2020 ). (2) Growth and sporulation of WT Beluga was tested in seven different biphasic media.…”

Section: Methodsmentioning

confidence: 99%

“…In part 1 of the study, we constructed sporulation mutants of C. botulinum Beluga using a novel CRISPR-Cas9 bookmark approach ( Seys et al, 2020 ). All PCR reactions for the cloning procedures were performed using the KOD Hot-Start high fidelity DNA polymerase (Merck).…”

Section: Methodsmentioning

confidence: 99%

“…Here we took the opportunity to exemplify CRISPR-Cas9 “bookmark” technology, a recently proposed concept ( Seys et al, 2020 ) for gold standard complementation studies, which exploits CRISPR-Cas9 to replace the mutant allele in situ with a wild type (WT) allele. Moreover, we established a novel medium for the characterization of spore formation in C. botulinum Group II strains and used it to determine the suitability of a previously described CRISPR-Cas9 system ( Ingle et al, 2019 ) for spore mutant generation in these strains.…”

Section: Introductionmentioning

confidence: 99%

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CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

et al. 2021

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The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt “bookmark” sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and “bookmark”-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique “watermark” through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

et al. 2021

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“…For future complementation studies, the 24 nt bookmark sequence is selected against to integrate the wildtype gene at its original location The pyrE gene in C. ljungdahlii was replaced with 9 consecutive bookmark sequences. All 9 bookmark sequences allowed complementation with the pyrE wildtype gene [ 264 ] Editing of single nucleotides in genome CRISPR-targeted base editing via deamination A combination of nuclease deactivated Cas9 with activation-induced cytidine deaminase is applied for cytosine to thymine substitution without DNA cleavage Premature stop codons were introduced into genes related to the formation of acetate ( pta ) and ethanol (adhE1, adhE2, aor1, aor2) in C. ljungdahlii [ 320 ] Editing of single nucleotides in genome CRISPR nickase assisted base editing via deamination A fusion of cytidine deaminase, CRISPR-Cas9 D10A nickase and uracil DNA glycosylase inhibitor (UGI) is used for base-pair substitutions of C∙G to A∙T Mutations were introduced into the pyrE, xylR, Spo0A and araR gene of C. beijerinckii [ 163 ] …”

Section: Systems Biology and Genetic Engineeringmentioning

confidence: 99%

Towards continuous industrial bioprocessing with solventogenic and acetogenic clostridia: challenges, progress and perspectives

Vees

Neuendorf

Pflügl

2020

Journal of Industrial Microbiology and Biotechnology

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The sustainable production of solvents from above ground carbon is highly desired. Several clostridia naturally produce solvents and use a variety of renewable and waste-derived substrates such as lignocellulosic biomass and gas mixtures containing H2/CO2 or CO. To enable economically viable production of solvents and biofuels such as ethanol and butanol, the high productivity of continuous bioprocesses is needed. While the first industrial-scale gas fermentation facility operates continuously, the acetone–butanol–ethanol (ABE) fermentation is traditionally operated in batch mode. This review highlights the benefits of continuous bioprocessing for solvent production and underlines the progress made towards its establishment. Based on metabolic capabilities of solvent producing clostridia, we discuss recent advances in systems-level understanding and genome engineering. On the process side, we focus on innovative fermentation methods and integrated product recovery to overcome the limitations of the classical one-stage chemostat and give an overview of the current industrial bioproduction of solvents.

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“…Similarly, several well-known inducible promoters were screened for Cas9 expression in E. limosum , and the anhydrotetracycline-inducible promoter was adopted for the CRISPR-Cas9 system in E. limosum [ 44 ]. Following its initial exemplification in several clostridial species [ 80 ], a synthetic, theophylline responsive riboswitch has also been exploited for Cas9-based mutant generation and complementation studies in C. autoethanogenum [ 81 ]. For the latter, unique 24-nucleotide “bookmark” sequences were incorporated into the mutant allele that acted as guide RNA targets during subsequent CRISPR-Cas9-mediated replacement with the complementing wildtype allele.…”

Section: Development Of Genetic Manipulation Tools In Acetogensmentioning

confidence: 99%

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals

Jin

Bae

Song

et al. 2020

IJMS

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Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.

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A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

Cited by 13 publications

References 21 publications

CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

Towards continuous industrial bioprocessing with solventogenic and acetogenic clostridia: challenges, progress and perspectives

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals

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