Reprogramming acetogenic bacteria with CRISPR-targeted base editingviadeamination

Abstract

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.

Development of a base editor for protein evolution via in situ mutation in vivo

“…Cas9 variants, which mainly include Cas9 nickase (D10A, usually denoted nCas9) and catalytically inactive Cas9 (D10A/H840A, usually denoted dCas9), fuse with deaminases to induce C:G to T:A or A:T to G:C conversion ( 19 ). Numerous tools derived from BEs have been successfully applied in gene insertions, gene deletions, and point mutations in various bacterial ( 20–24 ), mammalian ( 25–27 ) and plant cells ( 28–30 ). Due to the diverse genome environments in diverse cells and host-dependent factors of some tools, one tool is usually applied for one kind of cell.…”

Base Editing for Reprogramming Cyanobacterium Synechococcus elongatus

“…The mixed populations of edited strains seem to be a common issue for base editing, which have been observed in different microbes, such as C. ljungdahlii, Clostridium beijerinckii , and Agrobacterium spp. (29, 31, 36). Moreover, we achieved, to our best knowledge, the first multiplex genome editing in the species of Synechococcus , and two distant loci with completely different protospacers were edited simultaneously (Fig.…”

“…Base editing has been successfully adapted in bacteria, such as Streptomyces griseofuscus, Agrobacterium rhizogenes, Pseudomonas putida and Clostridium ljungdahlii (28)(29)(30)(31). To be noticed, Xia et al (2020) reported the potential of base editing in metabolic engineering of Clostridium ljungdahlii via a genome-scale interrogation, and demonstrated that base editing could overcome the challenges of applying CRISPR-Casbased genome-editing in bacteria, including the toxicity. Therefore, base editing may also shed light on the development of genome-editing tools for cyanobacteria.…”

Tailoring CRISPR-Cas Immunity for the Degradation of Antibiotic Resistance Genes

“…), and inverse PCRs were following previously established protocols in the lab. 28 Plasmid extraction and purification were conducted using commercial kits from Tiangen Biotech. PCRs were performed with PrimeSTAR Max DNA Polymerase from Takara Bio.…”