The CRISPR toolbox for the gram-positive model bacterium <i>Bacillus subtilis</i>

Zocca, Vitória Fernanda Bertolazzi; Corrêa, Graciely Gomes; Lins, Milca Rachel da Costa Ribeiro; Jesus, Victor Nunes de; Tavares, Leonardo Ferro; Amorim, Laura Araujo da Silva; Kundlatsch, Guilherme Engelberto; Pedrolli, Danielle Biscaro

doi:10.1080/07388551.2021.1983516

Cited by 15 publications

(14 citation statements)

References 70 publications

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“…In B. subtilis , different types of CRISPR‐Cas9‐based systems have been established according to the deployment strategies of Cas9, sgRNA and editing templates. Hong et al ( 2018 ) briefly reviewed a similar concept, and the employed plasmids and details were updated by Zocca et al ( 2021 ). With several options available, it is still critical to pay attention to specific experimental goals to choose the most suitable strategy.…”

Section: Effects Of Crispr Components On Genome Engineering Efficienc...mentioning

confidence: 99%

“…When describing the different CRISPR systems, the editing efficiencies of gene knockout, knockin or mutation were lightly sketched by Zocca et al ( 2021 ). In this review, we analyse the possible influencing factors and compare the editing efficiencies of different engineering methods.…”

Section: The Efficiency Of Different Gene Editing and Possible Influe...mentioning

confidence: 99%

“…These authors also thoroughly discussed the engineering of B. subtilis to produce industrially relevant compounds using CRISPR tools. The advancements in other Bacillus species and bacteriophages were only briefly mentioned (Zocca et al, 2021 ). This review emphasizes the effects of each CRISPR component on genome editing efficiency and discusses the plasmid‐curing strategies aiming for iterative engineering.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Effects Of Crispr Components On Genome Engineering Efficienc...mentioning

confidence: 99%

Section: The Efficiency Of Different Gene Editing and Possible Influe...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…Bacillus subtilis has been utilized extensively in commercial enzyme production. Bacillus subtilis possesses a number of advantages, including nonpathogenicity, suitability for large-scale fermentation, and a diverse toolkit for genetic manipulation [ 16 , 17 , 18 , 19 , 20 ]. In addition, various signal peptides and promoters can be used to achieve effective heterologous protein expression in Bacillus subtilis [ 16 , 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

Enhanced Extracellular Production and Characterization of Sucrose Isomerase in Bacillus subtilis with Optimized Signal Peptides

Guo

Jiang

et al. 2022

Foods

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Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, which is an important functional sugar widely used in the food industry. However, the lack of safe and efficient expression systems for recombinant SIase has impeded its production and application. In this study, enhanced expression of a SIase from Klebsiella sp. LX3 (referred to as KsLX3-SIase) was achieved in Bacillus subtilis WB800N, by optimizing the signal peptides. First, 13 candidate signal peptides were selected using a semi-rational approach, and their effects on KsLX3-SIase secretion were compared. The signal peptide WapA was most efficient in directing the secretion of KsLX3-SIase into the culture medium, producing a specific activity of 23.0 U/mL, as demonstrated by shake flask culture. Using a fed-batch strategy, the activity of KsLX3-SIase in the culture medium was increased to 125.0 U/mL in a 5-L fermentor. Finally, the expressed KsLX3-SIase was purified and was found to have maximum activity at 45 °C and pH 5.5. Its Km for sucrose was 267.6 ± 18.6 mmol/L, and its kcat/Km was 10.1 ± 0.2 s−1mM−1. These findings demonstrated an efficient expression of SIase in B. subtilis, and this is thought to be the highest level of SIase produced in a food-grade bacteria to date.

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“…Our current knowledge about CRISPR-mediated genome editing in bacteria mainly come from a number of human pathogens as well as industrial and laboratory strains, such as Escherichia coli [8–10], Pseudomonas [11, 12], Mycobacterium [13], Bacillus [14, 15], Corynebacterium [16], Clostridium [17], Streptomyces [18], etc . Generally speaking, the type II CRISPR/Cas9 system[19] is the most widely-used heterologous genome editor in various bacterial strains due to its robust nuclease activity and broad compatibility with other functional proteins (i.e.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

Yan¹,

Wang²,

Zhang³

et al. 2022

Preprint

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CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterogenous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion and plasmid cure in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 Xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.

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The CRISPR toolbox for the gram-positive model bacterium Bacillus subtilis

Cited by 15 publications

References 70 publications

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

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

Enhanced Extracellular Production and Characterization of Sucrose Isomerase in Bacillus subtilis with Optimized Signal Peptides

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

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