Unexpected evolutionary benefit to phages imparted by bacterial CRISPR-Cas9

Pan, Tao; Wu, Xianghua; Rao, Venigalla B.

doi:10.1126/sciadv.aar4134

Cited by 53 publications

(71 citation statements)

References 38 publications

(65 reference statements)

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“…Insertion sequences (ISs) and bacteriophages contribute actively to bacterial evolution by integrating and exciting from the genome. In certain conditions, they provide new genetic properties such as virulence factors and antibiotic resistance (41–45). In K. rhizophila no such observations for IS elements with respect to virulence and antibiotic determinates could be done, also the bacteriophage did not harbour important genes related to the bacterial cellular functions.…”

Section: Discussionmentioning

confidence: 99%

Functional and Comparative Genomics of Niche-Specific Adapted ActinomycetesKocuria rhizophilaStrain D2 Isolated from Healthy Human Gut

Ghattargi

Nimonkar

Sape³

et al. 2018

Preprint

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19Incidences of infection and occurrence of Kocuria rhizophila in human gut are prominent but 20 certainly no reports on the species ability to withstand human gastrointestinal dynamics. Kocuria 21 rhizophila strain D2 isolated from healthy human gut was comprehensively characterized. The 22 functional analysis revealed the ability to produce various gastric enzymes and sensitive to major 23 clinical antibiotics. It also exhibited tolerance to acidic pH and bile salts. Strain D2 displayed 24 bile-salt hydrolytic (BSH) activity, strong cell surface traits such as hydrophobicity, auto-25 aggregation capacity and adherence to human HT-29 cell line. Prominently, it showed no 26 hemolytic activity and was susceptible to the human serum. Exploration of the genome led to the 27 discovery of the genes for the above said properties and has ability to produce various essential 28 amino acids and vitamins. Further, comparative genomics have identified core, accessory and 29 unique genetic features. The core genome has given insights into the phylogeny while the 30 accessory and unique genes has led to the identification of niche specific genes. Bacteriophage, 31 virulence factors and biofilm formation genes were absent with this species. Housing CRISPR 32 and antibiotic resistance gene was strain specific. The integrated approach of functional, genomic 33 and comparative analysis denotes the niche specific adaption to gut dynamics of strain D2. 34Moreover the study has comprehensively characterized genome sequence of each strain to know 35 the genetic difference and intern recognize the effects of on phenotype and functionality 36 complexity. The evolutionary relationship among strains along and adaptation strategies has been 37 included in this study. 38Significance: Reports of Kocuria rhizophila isolation from various sources have been reported 40 but the few disease outbreaks in humans and fishes have been prominent, but no supportive 41 3 evidence about the survival ability of Kocuria spp. within human GIT. Here, we report the gut 42 adaption potential of K. rhizophila strain D2 by functional and genomic analysis. Further; 43 comparative genomics reveals this adaption to be strain specific (Gluten degradation). Genetic 44 difference, evolutionary relationship and adaptation strategies have been including in this study. 45 48 positive, non-hemolytic, non-endospore-forming, non-motile and can grow at different oxygen 49 levels (aerobic, facultative and anaerobic) (1, 2). Presence of galactosamine and glucosamine 50 (amino sugars) as the main component of the cell wall, differ them from other members of the 51class Actinobacteria (2). This genus is normally inhabitants of dust, soil, water and food and in 52 humans colonizes skin, mucosa, oropharynx and gastrointestinal tract (GIT) (3-5). 53K. rhizophila was isolated from the rhizoplane of the narrow-leaved cattail (Typha angustifolia). 54 in 1999 (6). The strain ATCC 9341 was reclassified as K. rhizophila from Micrococcus luteus 55 (7). It has been isolated from viz. c...

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Section: Discussionmentioning

confidence: 99%

Functional and Comparative Genomics of Niche-Specific Adapted ActinomycetesKocuria rhizophilaStrain D2 Isolated from Healthy Human Gut

Ghattargi

Nimonkar

Sape³

et al. 2018

Preprint

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“…Off-target, sequence-dependent nicking or non-specific nicking have been recently reported for several other Cas effector proteins (McMahon et al, 2020;Murugan et al, 2020;Yan et al, 2019). Here, target sequence-dependent nicking activity may help to slow down phage replication and fight against phages that develop mutations in the target region (McMahon et al, 2020;Tao et al, 2018). Further studies are required to demonstrate the benefits of target sequence-dependent nicking activity in vivo.…”

Section: Discussionmentioning

confidence: 95%

High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

Murugan

Seetharam

Severin

et al. 2020

Preprint

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Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wildtype (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high -throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and offtarget sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

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“…It was demonstrated that phages are one of the driving forces of bacterial evolution and vice versa (Koskella and Brockhurst, 2014; Tao et al, 2018). The infection of lytic phages will finally lead to the burst of host cells to release progeny phages.…”

Section: Discussionmentioning

confidence: 99%

Specific Integration of Temperate Phage Decreases the Pathogenicity of Host Bacteria

Chen

Yang

et al. 2020

Front. Cell. Infect. Microbiol.

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Temperate phages are considered as natural vectors for gene transmission among bacteria due to the ability to integrate their genomes into a host chromosome, therefore, affect the fitness and phenotype of host bacteria. Many virulence genes of pathogenic bacteria were identified in temperate phage genomes, supporting the concept that temperate phages play important roles in increasing the bacterial pathogenicity through delivery of the virulence genes. However, little is known about the roles of temperate phages in attenuation of bacterial virulence. Here, we report a novel Bordetella bronchiseptica temperate phage, vB_BbrS_PHB09 (PHB09), which has a 42,129-bp dsDNA genome with a G+C content of 62.8%. Phylogenetic analysis based on large terminase subunit indicated that phage PHB09 represented a new member of the family Siphoviridae. The genome of PHB09 contains genes encoding lysogen-associated proteins, including integrase and cI protein. The integration site of PHB09 is specifically located within a pilin gene of B. bronchiseptica. Importantly, we found that the integration of phage PHB09 significantly decreased the virulence of parental strain B. bronchiseptica Bb01 in mice, most likely through disruption the expression of pilin gene. Moreover, a single shot of the prophage bearing B. bronchiseptica strain completely protected mice against lethal challenge with wild-type virulent B. bronchiseptica, indicating the vaccine potential of lysogenized strain. Our findings not only indicate the complicated roles of temperate phages in bacterial virulence other than simple delivery of virulent genes but also provide a potential strategy for developing bacterial vaccines.

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Unexpected evolutionary benefit to phages imparted by bacterial CRISPR-Cas9

Abstract: Phages show an elevated mutation rate and remarkably rapid evolution when attacked by the bacterial CRISPR/Cas system.

Cited by 53 publications

References 38 publications

Functional and Comparative Genomics of Niche-Specific Adapted ActinomycetesKocuria rhizophilaStrain D2 Isolated from Healthy Human Gut

Functional and Comparative Genomics of Niche-Specific Adapted ActinomycetesKocuria rhizophilaStrain D2 Isolated from Healthy Human Gut

High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

Specific Integration of Temperate Phage Decreases the Pathogenicity of Host Bacteria

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