RiboCas: A Universal CRISPR-Based Editing Tool for <i>Clostridium</i>

Cañadas, Inés C.; Groothuis, Daphne; Zygouropoulou, Maria; Rodrigues, Raquel; Minton, Nigel P.

doi:10.1021/acssynbio.9b00075

Cited by 72 publications

(95 citation statements)

References 45 publications

(94 reference statements)

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“…In C. ljungdahlii transformation efficiencies were low due to constitutive expression of Cas9, which would only allow growth of colonies that had both taken up the plasmid and undergone successful genome editing (Huang et al, 2016). Coupling of an inducible riboswitch with Cas9 has shown improved genomic editing in other Clostridia (Cañadas et al, 2019). Switching the original thiolase promoter with the riboswitch inducible promoter (the 2-AP riboswitch linked with the Cthe_2638 promoter, pbuE, P = 8 bp) resulted in improved performance in E. coli and allowed comparable transformation efficiency of non-Cas9 plasmids, while we were unable to get successful transformants of the constitutive Cas9 construct targeting aor1.…”

Section: Creation and Characterization Of Pta Derivative Strainsmentioning

confidence: 85%

The Metabolism of Clostridium ljungdahlii in Phosphotransacetylase Negative Strains and Development of an Ethanologenic Strain

Humphreys

Jack

et al. 2020

Front. Bioeng. Biotechnol.

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The sustainable production of chemicals from non-petrochemical sources is one of the greatest challenges of our time. CO 2 release from industrial activity is not environmentally friendly yet provides an inexpensive feedstock for chemical production. One means of addressing this problem is using acetogenic bacteria to produce chemicals from CO 2 , waste streams, or renewable resources. Acetogens are attractive hosts for chemical production for many reasons: they can utilize a variety of feedstocks that are renewable or currently waste streams, can capture waste carbon sources and covert them to products, and can produce a variety of chemicals with greater carbon efficiency over traditional fermentation technologies. Here we investigated the metabolism of Clostridium ljungdahlii, a model acetogen, to probe carbon and electron partitioning and understand what mechanisms drive product formation in this organism. We utilized CRISPR/Cas9 and an inducible riboswitch to target enzymes involved in fermentation product formation. We focused on the genes encoding phosphotransacetylase (pta), aldehyde ferredoxin oxidoreductases (aor1 and aor2), and bifunctional alcohol/aldehyde dehydrogenases (adhE1 and adhE2) and performed growth studies under a variety of conditions to probe the role of those enzymes in the metabolism. Finally, we demonstrated a switch from acetogenic to ethanologenic metabolism by these manipulations, providing an engineered bacterium with greater application potential in biorefinery industry.

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Section: Creation and Characterization Of Pta Derivative Strainsmentioning

confidence: 85%

The Metabolism of Clostridium ljungdahlii in Phosphotransacetylase Negative Strains and Development of an Ethanologenic Strain

Humphreys

Jack

et al. 2020

Front. Bioeng. Biotechnol.

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“…2). Finely-graded dCas9 expression may require native, metal-sensitive promoters 53 , or addition of translational-level control such as riboswitches 54 .…”

Section: Discussionmentioning

confidence: 99%

Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes

et al. 2020

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Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactatesecreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.

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“…2). Finely-graded dCas9 expression may require native, metal-sensitive promoters 63,64 or addition of translational-level control such as riboswitches 65 .…”

Section: Discussionmentioning

confidence: 99%

Pooled CRISPRi screening of the cyanobacteriumSynechocystissp. PCC 6803 for enhanced growth, tolerance, and chemical production

Yao

Shabestary²,

Björk³

et al. 2019

Preprint

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We developed an inducible CRISPRi gene repression library in the cyanobacterium Synechocystis sp. PCC 6803, where all annotated genes are targeted for repression. We used the library to estimate gene fitness in multiple conditions. The library revealed several mutants with increased specific growth rates (up to 17%), and transcriptomics of these mutants revealed common upregulation of genes within photosynthetic electron flow. We challenged the library with L-lactate stress to find more tolerant mutants. Repression of the peroxiredoxin Bcp2 increased growth rate by 49% in the presence of 0.1 M L-lactate. Finally, the library was transformed into a L-lactate-secreting strain, and droplet microfluidics sorting of top producers enriched sgRNAs targeting nutrient assimilation, redox modulation, and cyclic-electron flow.Several clones showed increased productivity in batch cultivations (up to 75%). In some cases, tolerance or productivity was enhanced by partial repression of essential genes, which are difficult to access by transposon insertion.

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RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium

Cited by 72 publications

References 45 publications

The Metabolism of Clostridium ljungdahlii in Phosphotransacetylase Negative Strains and Development of an Ethanologenic Strain

The Metabolism of Clostridium ljungdahlii in Phosphotransacetylase Negative Strains and Development of an Ethanologenic Strain

Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes

Pooled CRISPRi screening of the cyanobacteriumSynechocystissp. PCC 6803 for enhanced growth, tolerance, and chemical production

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