Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli

Shelake, Rahul Mahadev; Pramanik, Dibyajyoti; Kim, Jae‐Yean

doi:10.1128/mbio.02296-22

Cited by 17 publications

(16 citation statements)

References 56 publications

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“…We found only one protospacer containing 14 nucleotides as the spacer of the gRNA for the SNV at ISM-RS04210 edited with gRNA04 (Figure S3), showing that this off-target event resulted from the CRISPR-Cas system targeted editing. These data demonstrated that most of the mutations were the products of random deamination, while still, part of the mutations resulted from CRISPR-Cas-mediated targeting which is in accordance with previous reports on Agrobacterium spp., S. coelicolor , S. elongatus , and Escherichia coli. , Overall, our base editing system is a reliable method for genetic manipulation with limited off-target events, but whole-genome sequencing may be necessary to identify undesired mutations in the genome.…”

Section: Resultsmentioning

confidence: 99%

“…These data demonstrated that most of the mutations were the products of random deamination, while still, part of the mutations resulted from CRISPR-Cas-mediated targeting which is in accordance with previous reports on Agrobacterium spp., 23 S. coelicolor, 24 S. elongatus, 26 and Escherichia coli. 22,28 Overall, our base editing system is a reliable method for genetic manipulation with limited off-target events, but whole-genome sequencing may be necessary to identify undesired mutations in the genome.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Developing a Base Editing System for Marine Roseobacter Clade Bacteria

et al. 2023

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The Roseobacter clade bacteria are of great significance in marine ecology and biogeochemical cycles, and they are potential microbial chassis for marine synthetic biology due to their versatile metabolic capabilities. Here, we adapted a CRISPR-Cas-based system, base editing, with the combination of nuclease-deactivated Cas9 and deaminase for Roseobacter clade bacteria. Taking the model roseobacter Roseovarius nubinhibens as an example, we achieved precise and efficient genome editing at single-nucleotide resolution without generating double-strand breaks or requesting donor DNAs. Since R. nubinhibens can metabolize aromatic compounds, we interrogated the key genes in the β-ketoadipate pathway with our base editing system via the introduction of premature STOP codons. The essentiality of these genes was demonstrated, and for the first time, we determined PcaQ as a transcription activator experimentally. This is the first report of CRISPR-Cas-based genome editing in the entire clade of Roseobacter bacteria. We believe that our work provides a paradigm for interrogating marine ecology and biogeochemistry with direct genotype-and-phenotype linkages and potentially opens a new avenue for the synthetic biology of marine Roseobacter bacteria.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Developing a Base Editing System for Marine Roseobacter Clade Bacteria

et al. 2023

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“…Base editors facilitate nucleotide substitutions within the editing window through the action of adenine or cytosine deaminases. Efforts to increase editing efficiency have continued through the utilization of deaminase variants and Cas9 variants obtained via laboratory evolution. − Recently, dual base editors have been used to perform both adenine and cytosine conversions. − Due to their lack of causing DSBs, base editors exhibit low cytotoxicity and offer the advantage of not requiring donor template DNA, thus making them widely used in various microbes …”

Section: Crispr-cas-mediated Genome Editing Methodsmentioning

confidence: 99%

Multiplex CRISPR-Cas Genome Editing: Next-Generation Microbial Strain Engineering

Lim,

Lee

2024

J. Agric. Food Chem.

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Genome editing is a crucial technology for obtaining desired phenotypes in a variety of species, ranging from microbes to plants, animals, and humans. With the advent of CRISPR-Cas technology, it has become possible to edit the intended sequence by modifying the target recognition sequence in guide RNA (gRNA). By expressing multiple gRNAs simultaneously, it is possible to edit multiple targets at the same time, allowing for the simultaneous introduction of various functions into the cell. This can significantly reduce the time and cost of obtaining engineered microbial strains for specific traits. In this review, we investigate the resolution of multiplex genome editing and its application in engineering microorganisms, including bacteria and yeast. Furthermore, we examine how recent advancements in artificial intelligence technology could assist in microbial genome editing and engineering. Based on these insights, we present our perspectives on the future evolution and potential impact of multiplex genome editing technologies in the agriculture and food industry.

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“…Given the requirement for microbial dual-BE tools, researchers subsequently developed an ACBE (ABE8e-CDA-nCas9) system applied in Bacillus subtilis and an iACBE (evoCDA1-ABE9e-nCas) system in E. coli, respectively. , Notably, the efficiencies of ABE8e-CDA-nCas9 and iACBE were both up to 100%, which exhibited their great potential for microbial genome editing.…”

Section: Constructions Of Be-based Microbial Gene-editing Toolsmentioning

confidence: 99%

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications

Qin

Zhang

et al. 2023

ACS Synth. Biol.

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Base editors (BE) based on CRISPR systems are practical gene-editing tools which continue to drive frontier advances of life sciences. BEs are able to efficiently induce point mutations at target sites without double-stranded DNA cleavage. Hence, they are widely employed in the fields of microbial genome engineering. As applications of BEs continue to expand, the demands for base-editing efficiency, fidelity, and versatility are also on the rise. In recent years, a series of optimization strategies for BEs have been developed. By engineering the core components of BEs or adopting different assembly methods, the performance of BEs has been well optimized. Moreover, series of newly established BEs have significantly expanded the base-editing toolsets. In this Review, we will summarize the current efforts for BE optimization, introduce several novel BEs with versatility, and look forward to the broadened applications for industrial microorganisms.

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Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli

Cited by 17 publications

References 56 publications

Developing a Base Editing System for Marine Roseobacter Clade Bacteria

Developing a Base Editing System for Marine Roseobacter Clade Bacteria

Multiplex CRISPR-Cas Genome Editing: Next-Generation Microbial Strain Engineering

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications

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