Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing

Hamilton, Thomas A.; Pellegrino, Gregory M.; Therrien, Jasmine A.; Ham, Dalton T.; Bartlett, Peter C.; Karas, Bogumil J.; Gloor, Gregory B.; Edgell, David R.

doi:10.1038/s41467-019-12448-3

Cited by 98 publications

(119 citation statements)

References 51 publications

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“…Among these, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas3and Cas9-encoding phages provide a means to combat such threats by selectively killing AMR bacteria 6 . The CRISPR-Cas3 and CRISPR-Cas9 genome-editing constructs, which were designed to target AMR genes, were delivered into bacteria by packaging them into phages [7][8][9][10][11] to achieve AMR gene-specific bacterial killing [7][8][9][10][11][12] and prevent the spread of AMR genes 7 . However, because DNA cleavage of plasmid DNA does not result in bacterial death, at least not in the absence of other confounding variables, such as a toxin-antitoxin system, this strategy is ineffective in targeting bacteria with plasmid-borne AMR genes 9 .…”

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confidence: 99%

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

et al. 2020

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The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed Cap-sidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

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confidence: 99%

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

et al. 2020

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“…2). It is widely observed that Cas9 and its mutant dCas9 may exhibit off-target activity and/or sequence-specific toxicity in target organisms such as E. coli (Cui et al 2018;Hamilton et al 2019) and clostridia (Xu et al 2015). Previously, we observed cloning difficulties with plasmid piCas in E. coli DH5α which could be overcome by cloning of Lac repressor operator sequence (lacO) at the transcriptional starting site of the employed m2p promoter (Schultenkämper et al 2019).…”

Section: Discussionmentioning

confidence: 99%

CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5

Brito

Schultenkämper

Passaglia

et al. 2020

Appl Microbiol Biotechnol

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Gene repression using the endonucleolytically deactivated dCas9 protein and sgRNAs (CRISPR interference or CRISPRi) is a useful approach to study gene functions. Here, we established CRISPRi in Paenibacillus sonchi genomovar Riograndensis SBR5, a plant growth promoting bacterium. CRISPRi system with sgRNAs targeting SBR5 endogenous genes spo0A, yaaT and ydjJ and plasmid-borne gfpUV was constructed and analyzed. Flow cytometry analysis revealed a significant decrease of reporter protein GFPUV signal in P. sonchi strains expressing gfpUV sgRNA in comparison with non-targeting controls. CRISPRi-based repression of chromosomal genes for regulation of sporulation spo0A and yaaT decreased sporulation and increased biofilm formation in SBR5. Repression of the sorbitol catabolic gene ydjJ revealed decreased specific activity of YdjJ in crude cell extracts and reduced biomass formation from sorbitol in growth experiments. Our work on CRISPRi-based gene repression serves as basis for gene function studies of the plant growth promoter P. sonchi SBR5. To our knowledge, the present study presents the first tool for gene repression established in Paenibacillus species. Key points • CRISPRi toward gene repression was applied for the first time in Paenibacillus. • CRISPRi of spo0A and yaaT depleted spores and increased biofilms in SBR5. • CRISPRi-based ydjJ repression decreased specific activity of sorbitol dehydrogenase.

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“…102 Conjugative plasmids that encode and promote biofilm formation could increase rates of conjugative plasmid transfer due to the enhanced cell-to-cell contact, 103 which may be suited for delivery of molecular tools such as CRISPR nucleases for modulating composition of microbial communities 104,106 that many of them exist as biofilms. In a study by Hamilton et al, 107 they developed a cis-conjugative system that the plasmid encodes both CRISPR nuclease and conjugative machinery. They designed 65 total sgRNA, which target 38, 23, and 4 essential genes, non-essential genes and genes with unresolved phenotypes.…”

Section: Crispr-cas System Neutralizing Antibiotic-resistant Genesmentioning

confidence: 99%

<p>How CRISPR-Cas System Could Be Used to Combat Antimicrobial Resistance</p>

Gholizadeh¹,

Köse²,

Dao³

et al. 2020

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109

101

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Rapid emergence of antibiotic-resistant bacteria has made it harder for us to combat infectious diseases and to develop new antibiotics. The clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR-Cas) system, as a bacterial adaptive immune system, is recognized as one of the new strategies for controlling antibioticresistant strains. The programmable Cas nuclease of this system used against bacterial genomic sequences could be lethal or could help reduce resistance of bacteria to antibiotics. Therefore, this study aims to review using the CRISPR-Cas system to promote sensitizing bacteria to antibiotics. We envision that CRISPR-Cas approaches may open novel ways for the development of smart antibiotics, which could eliminate multidrug-resistant (MDR) pathogens and differentiate between beneficial and pathogenic microorganisms. These systems can be exploited to quantitatively and selectively eliminate individual bacterial strains based on a sequence-specific manner, creating opportunities in the treatment of MDR infections, the study of microbial consortia, and the control of industrial fermentation.

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Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing

Cited by 98 publications

References 51 publications

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5

<p>How CRISPR-Cas System Could Be Used to Combat Antimicrobial Resistance</p>

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