Chances and limitations when uncovering essential and non‐essential genes of <scp><i>Bacillus subtilis</i></scp> phages with <scp>CRISPR‐Cas9</scp>

Kohm, Katharina; Basu, Syamantak; Nawaz, Muhammad M; Hertel, Robert

doi:10.1111/1758-2229.13005

Cited by 6 publications

(3 citation statements)

References 40 publications

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“…B. subtilis strains were made competent as described by Anagnostopoulos and Spizizen (Anagnostopoulos and Spizizen, 1961) with modifications (Kohm et al ., 2021). B. subtilis mutants were created either through the transformation of the recipient stain with chromosomal DNA, a specific plasmid or PCR product.…”

Section: Methodsmentioning

confidence: 99%

The Bacillus phage SPβ and its relatives: a temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

Kohm

Floccari

Lutz

et al. 2022

Environmental Microbiology

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The Bacillus phage SPβ has been known for about 50 years, but only a few strains are available. We isolated four new wild-type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPβ-like phages. Sequence data revealed the genome replication strategy and the genome packaging mode of SPβ-like phages. We extracted 55 SPβlike prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resemble four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPβ-like prophages consists of 38 proteins. The integration cassette proved to be not conserved, even though, present in all strains. It consists of distinct integrases. Analysis of SPβ transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

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Section: Methodsmentioning

confidence: 99%

The Bacillus phage SPβ and its relatives: a temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

Kohm

Floccari

Lutz

et al. 2022

Environmental Microbiology

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“…This can happen if the repair is incomplete, or if the repair results in a frame shift mutation that causes the gene to produce a nonfunctional protein [48]. Some limitations, like low survival rates of B. subtilis phages after Cas9 attack, were observed when using vector pRH030 [49]. It is possible that the large size of hp1 may have caused Cas9-induced indels to affect not only hp1, but also other nearby genes, disrupting cellular processes such as cell cycle, DNA replication, and DNA repair [50].…”

Section: Limitations Of Type II Crispr/cas System For Precision Super...mentioning

confidence: 99%

Application of CRISPR-Cas System to Mitigate Superbug Infections

Rabaan,

Al Fares,

Almaghaslah

et al. 2023

Microorganisms

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Multidrug resistance in bacterial strains known as superbugs is estimated to cause fatal infections worldwide. Migration and urbanization have resulted in overcrowding and inadequate sanitation, contributing to a high risk of superbug infections within and between different communities. The CRISPR-Cas system, mainly type II, has been projected as a robust tool to precisely edit drug-resistant bacterial genomes to combat antibiotic-resistant bacterial strains effectively. To entirely opt for its potential, advanced development in the CRISPR-Cas system is needed to reduce toxicity and promote efficacy in gene-editing applications. This might involve base-editing techniques used to produce point mutations. These methods employ designed Cas9 variations, such as the adenine base editor (ABE) and the cytidine base editor (CBE), to directly edit single base pairs without causing DSBs. The CBE and ABE could change a target base pair into a different one (for example, G-C to A-T or C-G to A-T). In this review, we addressed the limitations of the CRISPR/Cas system and explored strategies for circumventing these limitations by applying diverse base-editing techniques. Furthermore, we also discussed recent research showcasing the ability of base editors to eliminate drug-resistant microbes.

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“…This vector can achieve constitutive expression of Cas9 and lead to inactivation of viral attackers and enhanced mutagenic efficiency of phages (Otte et al, 2020 ). Consequently, this Cas9‐based mutagenesis vector pRH030 was used as a genetic tool on B. subtilis model phage Goe1 to specifically disrupt all phage genes to verify essential or non‐essential genes for phage survival (Kohm et al, 2021 ).…”

Section: Insight Into Bacillus –Phages Relations W...mentioning

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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Chances and limitations when uncovering essential and non‐essential genes of Bacillus subtilis phages with CRISPR‐Cas9

Cited by 6 publications

References 40 publications

The Bacillus phage SPβ and its relatives: a temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

The Bacillus phage SPβ and its relatives: a temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

Application of CRISPR-Cas System to Mitigate Superbug Infections

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

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