Modulation of Cas9 level for efficient CRISPR-Cas9-mediated chromosomal and plasmid gene deletion in Bacillus thuringiensis

Soonsanga, Sumarin; Luxananil, Plearnpis; Promdonkoy, Boonhiang

doi:10.1007/s10529-020-02809-0

Cited by 13 publications

(4 citation statements)

References 19 publications

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“…In CRISPR-based genome editing, it is imperative that the expression of the elements comprising a CRISPR system (i.e., the cas gene and small guide RNA) be strictly controlled since high expression levels of Cas proteins are often toxic in a number of bacterial species [ 28 – 30 ]. Additionally, control of gene expression has been proven critical to obtaining the desired phenotype (e.g., high productivity of target metabolite) in metabolic engineering and synthetic biology studies [ 31 , 32 ].…”

Section: Resultsmentioning

confidence: 99%

Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP

Kwon,

Lee,

Kwon

et al. 2024

Microb Cell Fact

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Background Clostridium sp. AWRP (AWRP) is a novel acetogenic bacterium isolated under high partial pressure of carbon monoxide (CO) and can be one of promising candidates for alcohol production from carbon oxides. Compared to model strains such as C. ljungdahlii and C. autoethanogenum, however, genetic manipulation of AWRP has not been established, preventing studies on its physiological characteristics and metabolic engineering. Results We were able to demonstrate the genetic domestication of AWRP, including transformation of shuttle plasmids, promoter characterization, and genome editing. From the conjugation experiment with E. coli S17-1, among the four replicons tested (pCB102, pAMβ1, pIP404, and pIM13), three replicated in AWRP but pCB102 was the only one that could be transferred by electroporation. DNA methylation in E. coli significantly influenced transformation efficiencies in AWRP: the highest transformation efficiencies (102–103 CFU/µg) were achieved with unmethylated plasmid DNA. Determination of strengths of several clostridial promoters enabled the establishment of a CRISPR/Cas12a genome editing system based on Acidaminococcus sp. BV3L6 cas12a gene; interestingly, the commonly used CRISPR/Cas9 system did not work in AWRP, although it expressed the weakest promoter (C. acetobutylicum Pptb) tested. This system was successfully employed for the single gene deletion (xylB and pyrE) and double deletion of two prophage gene clusters. Conclusions The presented genome editing system allowed us to achieve several genome manipulations, including double deletion of two large prophage groups. The genetic toolbox developed in this study will offer a chance for deeper studies on Clostridium sp. AWRP for syngas fermentation and carbon dioxide (CO2) sequestration.

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Section: Resultsmentioning

confidence: 99%

Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP

Kwon,

Lee,

Kwon

et al. 2024

Microb Cell Fact

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“…The transfer of "useful" insectotoxin genes from other economically important Bt strains to endophytic bacteria, as well as the maintenance of their consortiums, should contribute to the creation of new-generation biological agents based on them. At the same time, modern technologies for editing microbial genomes based on the CRISPRCas9 platform [168,175] can be proposed to disable the α-exotoxin and β-exotoxin synthesis of Bt.…”

Section: Improvement Of Insecticidal Properties Of Bacterial Strainsmentioning

confidence: 99%

Plant-Associated Bacillus thuringiensis and Bacillus cereus: Inside Agents for Biocontrol and Genetic Recombination in Phytomicrobiome

Sorokan,

Gabdrakhmanova,

Kuramshina

et al. 2023

Plants

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Bacillus thuringiensis Berliner (Bt) and B. cereus sensu stricto Frankland and Frankland are closely related species of aerobic, spore-forming bacteria included in the B. cereus sensu lato group. This group is one of the most studied, but it remains also the most mysterious species of bacteria. Despite more than a century of research on the features of these ubiquitous bacteria, there are a lot of questionable issues related to their taxonomy, resistance to external influences, endophytic existence, their place in multidimensional relationships in the ecosystem, and many others. The review summarizes current data on the mutualistic relationships of Bt and B. cereus bacteria with plants, the structure of the phytomicrobiomes including Bt and B. cereus, and the abilities of plant-associated and endophytic strains to improve plant resistance to various environmental factors and its productivity. Key findings on the possibility of the use of Cry gene promoter for transcription of the target dsRNA and simultaneous release of pore-forming proteins and provocation of RNA-interference in pest organisms allow us to consider this group of microorganisms as unique tools of genetic engineering and biological control. This will open the prospects for the development and direct change of plant microbiomes, and possibly serve as the basis for the regulation of the entire agroecosystem.

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“…To further improve its ability to produce stable and functional crystal and vegetative insecticidal proteins at a large scale, precise gene engineering is required. The CRISPR‐Cas9 strategy has been successfully developed to delete genes in this species, in which double crossover recombination was rarely occurring (Soonsanga et al, 2020 ; Tan et al, 2019 ). Though the obtained transformant numbers were very low, the editing efficiency was still acceptable (12.5%–62.5%).…”

Section: Genome Engineering In Other Bacillus Speciesmentioning

confidence: 99%

Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages

Song

Jopkiewicz

et al. 2022

Journal of Applied Microbiology

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Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria‐based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up‐to‐date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful.

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Modulation of Cas9 level for efficient CRISPR-Cas9-mediated chromosomal and plasmid gene deletion in Bacillus thuringiensis

Cited by 13 publications

References 19 publications

Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP

Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP

Plant-Associated Bacillus thuringiensis and Bacillus cereus: Inside Agents for Biocontrol and Genetic Recombination in Phytomicrobiome

Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages

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