Genome editing of Clostridium autoethanogenum using CRISPR/Cas9

Nagaraju, Shilpa; Davies, Naomi; Walker, David M.; Köpke, Michael; Simpson, Séan D.

doi:10.1186/s13068-016-0638-3

Cited by 105 publications

(65 citation statements)

References 26 publications

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“…Codon-adapted luciferase genes for CFE were synthesized by IDT, cloned into the pJL1 plasmid using Gibson assembly and confirmed by Sanger sequencing by ACGT, Inc. Kanamycin (50 μg/ mL) was used to maintain pJL1-based plasmids. C. auto endogenous promoters of phosphotransacetylase-actetate kinase operon (pPta-Ack; CAETHG_RS16490), pyruvate:formate oxidoreductase (pPFOR; CAETHG_RS14890) and Wood-Ljungdahl cluster (pWL; CAETHG_RS07860) were amplified from a plasmid where the respective sequences have been amplified from the genome and cloned into a pMTL82250 vector reporter plasmid (Nagaraju et al, 2016) and cloned in place of the T7 promoter region in the pJL1-LucCae construct using Gibson assembly and confirmed by Sanger sequencing by ACGT, Inc.…”

Section: Methodsmentioning

confidence: 99%

“…However, the current state-of-the-art strain engineering for clostridia remains a low-throughput, labor-intensive endeavor. Specific challenges include organism-specific genetic constraints (Daniell et al, 2015;Joseph et al, 2018;Liew et al, 2017Liew et al, , 2016Nagaraju et al, 2016), the requirement of an anaerobic environment, and, in case of acetogens, handling of gases. As a result, developments in clostridia biotechnology and basic knowledge of clostridia biology have lagged behind achievements in aerobic prokaryotic and eukaryotic biology.…”

Section: Introductionmentioning

confidence: 99%

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Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Krüger¹,

Mueller

Rybnicky³

et al. 2020

Preprint

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Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste.Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic parts, gene expression, and biosynthetic pathway performance in vitro before implementing them in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 µg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Krüger¹,

Mueller

Rybnicky³

et al. 2020

Preprint

Self Cite

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“…We designed a modularized system to enable fast generation of the base-editing plasmid series. The employed plasmid backbone, replicon for clostridia, antibiotic resistances markers, and the dCas9 protein have been separately demonstrated to be functional in various species in the order Clostridiales, including Acetobacterium woodii (dCas9 has not yet been validated) (31,32), Eubacterium limosum (13), and Clostridium autoethanogenum (14). Accordingly, the system could be easily generalized in acetogenic bacteria, which mainly belong to the order Clostridiales.…”

Section: An Expanded Synthetic Biology Toolkit For Acetogenic Bacteriamentioning

confidence: 99%

“…and Cas12a) have been established in C. ljungdahlii (10)(11)(12), Eubacterium limosum (13), and C. autoethanogenum (14). For these systems, first, the Cas protein (e.g., Cas9 from Streptococcus pyogenes) is targeted to a highly specific site on the genome by a guide RNA (gRNA).…”

Section: Introductionmentioning

confidence: 99%

Reprogramming acetogenic bacteria with CRISPR-targeted base editingviadeamination

Xia

Casini

Schulz

et al. 2020

Preprint

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Acetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-tothymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome.Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general. SignificanceAcetogenic bacteria metabolize one-carbon (C1) gases, such as industrial waste gases, to produce fuels and commodity chemicals. However, the lack of efficient genemanipulation approaches hampers faster progress in the application of acetogenic bacteria in biotechnology. We developed a CRISPR-targeted base-editing tool at a onenucleotide resolution for acetogenic bacteria. Our tool illustrates great potential in engineering other A-T-rich bacteria and links designed single-nucleotide variations with biotechnology. It provides unique advantages for engineering industrially relevant bacteria without creating genetically modified organisms (GMOs) under the legislation of many countries. This base-editing tool provides an example for adapting CRISPR-Cas systems in bacteria, especially those that are highly sensitive to heterologously expressed Cas proteins and have limited ability of receiving foreign DNA.

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“…For the cell-free work, the pJL1 plasmid (Addgene #69496) was used. The modular pMTL80000 plasmid system 60 along with acsA 45 , fdx 45 , pta 61 and pfor 62 promoters were used for the C. autoethanogenum plasmid expression.…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

In vitroprototyping and rapid optimization of biosynthetic enzymes for cellular design

Karim

Dudley

Juminaga

et al. 2019

Preprint

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Microbial cell factories offer an attractive approach for production of biobased products.Unfortunately, designing, building, and optimizing biosynthetic pathways remains a complex challenge, especially for industrially-relevant, non-model organisms. To address this challenge, we describe a platform for in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (iPROBE). In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE with two examples. First, we tested and ranked 54 different pathways for 3-hydroxybutyrate production, improving in vivo production in Clostridium by 20-fold to 14.63 ± 0.48 g/L and identifying a new biosynthetic route to (S)-(+)-1,3butanediol. Second, we used iPROBE and data-driven design to optimize a 6-step n-butanol pathway, increasing titers 4-fold across 205 pathways, and showed strong correlation between cell-free and cellular performance. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

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Genome editing of Clostridium autoethanogenum using CRISPR/Cas9

Cited by 105 publications

References 26 publications

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Reprogramming acetogenic bacteria with CRISPR-targeted base editingviadeamination

In vitroprototyping and rapid optimization of biosynthetic enzymes for cellular design

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