Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination

Acetogenic bacteria are rising in popularity as chassis microbes for 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 fun...

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Bibliographic Details
Published inACS synthetic biology Vol. 9; no. 8; pp. 2162 - 2171
Main Authors Xia, Peng-Fei, Casini, Isabella, Schulz, Sarah, Klask, Christian-Marco, Angenent, Largus T, Molitor, Bastian
Format Journal Article
LanguageEnglish
Published American Chemical Society 21.08.2020
Subjects
synthetic biology
CRISPR
acetogenic bacteria
base editing
Clostridium ljungdahlii
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Summary:Acetogenic bacteria are rising in popularity as chassis microbes for 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-to-thymine 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 and the potential for off-target events. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux toward 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.
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ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.0c00226