Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system

Kiro, Ruth; Shitrit, Dror; Qimron, Udi

doi:10.4161/rna.27766

Cited by 135 publications

(155 citation statements)

References 22 publications

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“…In BRED, bacterial cells inducibly expressing recombination functions are electroporated with a combination of phage DNA template and a targeting substrate. Moreover, CRISPR-Cas system is also exploited to edit the genome of phages by increasing recombination efficiencies (32) or used to counterselect nonedited phage genomes (33). In the present work, we modified the reported methods to generate Klebsiella bacteriophage mutants for further examination.…”

Section: Discussionmentioning

confidence: 99%

Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types

Pan

Lin

Chen

et al. 2017

J Virol

115

111

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The genome of the multihost bacteriophage ⌽K64-1, capable of infecting Klebsiella capsular types K1, K11, K21, K25, K30, K35, K64, and K69, as well as new capsular types KN4 and KN5, was analyzed and revealed that 11 genes (S1-1, S1-2, S1-3, S2-1, S2-2, S2-3, S2-4, S2-5, S2-6, S2-7, and S2-8) encode proteins with amino acid sequence similarity to tail fibers/spikes or lyases. S2-5 previously was shown to encode a K64 capsule depolymerase (K64dep). Specific capsule-degrading activities of an additional eight putative capsule depolymerases (S2-4 against K1, S1-1 against K11, S1-3 against K21, S2-2 against K25, S2-6 against K30/K69, S2-3 against K35, S1-2 against KN4, and S2-1 against KN5) was demonstrated by expression and purification of the recombinant proteins. Consistent with the capsular type-specific depolymerization activity of these gene products, phage mutants of S1-2, S2-2, S2-3, or S2-6 lost infectivity for KN4, K25, K35, or K30/K69, respectively, indicating that capsule depolymerase is crucial for infecting specific hosts. In conclusion, we identified nine functional capsule depolymerase-encoding genes in a bacteriophage and correlated activities of the gene products to all ten hosts of this phage, providing an example of type-specific host infection mechanisms in a multihost bacteriophage.IMPORTANCE We currently identified eight novel capsule depolymerases in a multihost Klebsiella bacteriophage and correlated the activities of the gene products to all hosts of this phage, providing an example of carriage of multiple depolymerases in a phage with a wide capsular type host spectrum. Moreover, we also established a recombineering system for modification of Klebsiella bacteriophage genomes and demonstrated the importance of capsule depolymerase for infecting specific hosts. Based on the powerful tool for modification of phage genome, further studies can be conducted to improve the understanding of mechanistic details of Klebsiella phage infection. Furthermore, the newly identified capsule depolymerases will be of great value for applications in capsular typing.KEYWORDS Klebsiella, bacteriophage, capsular type, capsule depolymerase, multiple host T he genus Klebsiella, especially the species Klebsiella pneumoniae, is an important human pathogen that causes a wide range of diseases, including both community and hospital-acquired infections. It is associated with septicemia, pneumonia, and urinary tract infections (1, 2) and also is responsible for a globally emerging disease, pyogenic liver abscess complicated with metastatic meningitis and endophthalmitis (3,4).Klebsiella spp. typically display a layer of thick, polysaccharide-based capsule on their surfaces. The expression of diverse capsule structure caused by different sugar compositions and linkages divide them into distinct serotypes. In addition, genetic

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Section: Discussionmentioning

confidence: 99%

Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types

Pan

Lin

Chen

et al. 2017

J Virol

115

111

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“…We determined the sequence requirements for system activity both by targeting divergent sequences between related phages and by assessing viruses that escape CRISPR-Cas-mediated resistance. Furthermore, in light of recent advances in engineering phages using CRISPR-Cas (28,29), we adapt this CRISPR-Cas system for efficient engineering of lytic V. cholerae phage genomes.…”

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confidence: 99%

Functional Analysis of Bacteriophage Immunity through a Type I-E CRISPR-Cas System in Vibrio cholerae and Its Application in Bacteriophage Genome Engineering

et al. 2016

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The classical and El Tor biotypes of Vibrio cholerae serogroup O1, the etiological agent of cholera, are responsible for the sixth and seventh (current) pandemics, respectively. A genomic island (GI), GI-24, previously identified in a classical biotype strain of V. cholerae, is predicted to encode clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins); however, experimental evidence in support of CRISPR activity in V. cholerae has not been documented. Here, we show that CRISPR-Cas is ubiquitous in strains of the classical biotype but excluded from strains of the El Tor biotype. We also provide in silico evidence to suggest that CRISPR-Cas actively contributes to phage resistance in classical strains. We demonstrate that transfer of GI-24 to V. cholerae El Tor via natural transformation enables CRISPR-Cas-mediated resistance to bacteriophage CP-T1 under laboratory conditions. To elucidate the sequence requirements of this type I-E CRISPR-Cas system, we engineered a plasmid-based system allowing the directed targeting of a region of interest. Through screening for phage mutants that escape CRISPR-Cas-mediated resistance, we show that CRISPR targets must be accompanied by a 3= TT protospacer-adjacent motif (PAM) for efficient interference. Finally, we demonstrate that efficient editing of V. cholerae lytic phage genomes can be performed by simultaneously introducing an editing template that allows homologous recombination and escape from CRISPR-Cas targeting. IMPORTANCECholera, caused by the facultative pathogen Vibrio cholerae, remains a serious public health threat. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) provide prokaryotes with sequence-specific protection from invading nucleic acids, including bacteriophages. In this work, we show that one genomic feature differentiating sixth pandemic (classical biotype) strains from seventh pandemic (El Tor biotype) strains is the presence of a CRISPR-Cas system in the classical biotype. We demonstrate that the CRISPR-Cas system from a classical biotype strain can be transferred to a V. cholerae El Tor biotype strain and that it is functional in providing resistance to phage infection. Finally, we show that this CRISPR-Cas system can be used as an efficient tool for the editing of V. cholerae lytic phage genomes. Vibrio cholerae is a Gram-negative facultative pathogen that causes the acute diarrheal disease cholera. The current cholera pandemic, the seventh in recorded history, began in 1961 and is caused by V. cholerae O1 of the El Tor biotype (1). This pandemic has affected much of the developing world, including many countries in Asia and Africa and most recently in the Caribbean (2). The sixth cholera pandemic, however, was caused by V. cholerae O1 of the classical biotype. Classical biotype strains declined following the emergence of El Tor strains and are now believed to be extinct, after last being seen in 1990 in Bangladesh (3). The mechanisms underpinn...

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“…The mode of action of CRISPR-Cas systems comprises three main processes, namely, CRISPR adaptation, RNA biogenesis, and CRISPR-Cas interference, which are further reviewed by Westra et al (74) and Makarova et al (77).neering of the T7 phage genome by using the type I-E CRISPR-Cas system (Fig. 4) (79). Homologous recombination was first used to delete a nonessential T7 gene (gene 1.7).…”

Section: Crispr-cas-mediated Genome Engineeringmentioning

confidence: 99%

“…The phage population resulting from this reaction contained recombinant phages lacking gene 1.7 as well as nonrecombinant wild-type phages. In order to enrich for the desired recombinant phages, a CRISPR-based counterselection system was used (79). The recombinant phage lysate was plated on host bacteria carrying 3 plasmids encoding the components required for CRISPR-Cas activity: the targeting cascade complex, the cas3 degradation machinery, and the CRISPR spacer targeting gene 1.7.…”

Section: Crispr-cas-mediated Genome Engineeringmentioning

confidence: 99%

“…The result was selective cleavage of the nonrecombinant phage genomes, which contained gene 1.7, but not of the recombinant phage genomes, which lacked gene 1.7, thus inhibiting replication of the former phages and enriching for the latter phages. This method thus overcomes the issue of having to fish out a very small percentage of recombinant phages from a large pool of wild-type phages (79). Similarly, the Streptococcus thermophilus type II-A CRISPR-Cas system has been used to select for Streptococcus phage 2972 recombinants that have undergone point mutations, small and large DNA deletions, and gene replacements (80).…”

Section: Crispr-cas-mediated Genome Engineeringmentioning

confidence: 99%

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Genetically Engineered Phages: a Review of Advances over the Last Decade

Pires

Cleto

Sillankorva

et al. 2016

Microbiol Mol Biol Rev

315

275

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SUMMARYSoon after their discovery in the early 20th century, bacteriophages were recognized to have great potential as antimicrobial agents, a potential that has yet to be fully realized. The nascent field of phage therapy was adversely affected by inadequately controlled trials and the discovery of antibiotics. Although the study of phages as anti-infective agents slowed, phages played an important role in the development of molecular biology. In recent years, the increase in multidrug-resistant bacteria has renewed interest in the use of phages as antimicrobial agents. With the wide array of possibilities offered by genetic engineering, these bacterial viruses are being modified to precisely control and detect bacteria and to serve as new sources of antibacterials. In applications that go beyond their antimicrobial activity, phages are also being developed as vehicles for drug delivery and vaccines, as well as for the assembly of new materials. This review highlights advances in techniques used to engineer phages for all of these purposes and discusses existing challenges and opportunities for future work.

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Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system

Cited by 135 publications

References 22 publications

Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types

Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types

Functional Analysis of Bacteriophage Immunity through a Type I-E CRISPR-Cas System in Vibrio cholerae and Its Application in Bacteriophage Genome Engineering

Genetically Engineered Phages: a Review of Advances over the Last Decade

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