Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination

• DOI: 10.1021/acssynbio.0c00226
• Publication Date: 2020-07-01T22:38:26Z
• Authors: Peng-Fei Xia; Isabella Casini; Sarah Schulz; Christian-Marco K...; Largus T. Angenent; Bastian Molitor
• Keywords: Synthetic Biology

Acetogenic bacteria are rising in popularity as chassis microbes for biotechnology due to their capability of converting inorganic one-carbon (C1) gases organic chemicals. To fully uncover the potential acetogenic bacteria, synthetic biology tools imperative either engineer designed functions or interrogate physiology. Here, we report a genome-editing tool at one-nucleotide resolution, namely base editing, based on CRISPR-targeted deamination. This combines nuclease deactivated Cas9 with activation-induced cytidine deaminase enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which generally bottlenecks applying conventional CRISPR-Cas systems bacteria. We validated modularized base-editing model bacterium Clostridium ljungdahlii. The editing principles were investigated, an in-silico analysis revealed across genome off-target events. Moreover, genes related acetate ethanol production disrupted individually by installing premature STOP codons reprogram carbon flux toward improved production. resulted engineered C. ljungdahlii strains desired phenotypes stable genotypes. Our promotes application research provides blueprint upgrade CRISPR-Cas-based general.