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Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination.
ACS Synth Biol. 2020 Jul 16 [Online ahead of print]AS

Abstract

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.

Authors+Show Affiliations

Environmental Biotechnology Group, Center for Applied Geosciences, University of Tübingen, 72074 Tübingen, Germany.Environmental Biotechnology Group, Center for Applied Geosciences, University of Tübingen, 72074 Tübingen, Germany.Environmental Biotechnology Group, Center for Applied Geosciences, University of Tübingen, 72074 Tübingen, Germany.Environmental Biotechnology Group, Center for Applied Geosciences, University of Tübingen, 72074 Tübingen, Germany.Environmental Biotechnology Group, Center for Applied Geosciences, University of Tübingen, 72074 Tübingen, Germany. AG Angenent, Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany.Environmental Biotechnology Group, Center for Applied Geosciences, University of Tübingen, 72074 Tübingen, Germany.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32610012

Citation

Xia, Peng-Fei, et al. "Reprogramming Acetogenic Bacteria With CRISPR-Targeted Base Editing Via Deamination." ACS Synthetic Biology, 2020.
Xia PF, Casini I, Schulz S, et al. Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination. ACS Synth Biol. 2020.
Xia, P. F., Casini, I., Schulz, S., Klask, C. M., Angenent, L. T., & Molitor, B. (2020). Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination. ACS Synthetic Biology. https://doi.org/10.1021/acssynbio.0c00226
Xia PF, et al. Reprogramming Acetogenic Bacteria With CRISPR-Targeted Base Editing Via Deamination. ACS Synth Biol. 2020 Jul 16; PubMed PMID: 32610012.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination. AU - Xia,Peng-Fei, AU - Casini,Isabella, AU - Schulz,Sarah, AU - Klask,Christian-Marco, AU - Angenent,Largus T, AU - Molitor,Bastian, Y1 - 2020/07/16/ PY - 2020/7/2/pubmed PY - 2020/7/2/medline PY - 2020/7/2/entrez KW - CRISPR KW - Clostridium ljungdahlii KW - acetogenic bacteria KW - base editing KW - synthetic biology JF - ACS synthetic biology JO - ACS Synth Biol N2 - 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. SN - 2161-5063 UR - https://www.unboundmedicine.com/medline/citation/32610012/Reprogramming_acetogenic_bacteria_with_CRISPR-targeted_base_editing_via_deamination L2 - https://doi.org/10.1021/acssynbio.0c00226 DB - PRIME DP - Unbound Medicine ER -
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