A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

Seys, François M.; Rowe, Peter; Bolt, Edward L.; Humphreys, Christopher M.; Minton, Nigel P.

doi:10.20944/preprints202003.0321.v1

Cited by 4 publications

(2 citation statements)

References 17 publications

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“…The homology cassette consisted of sgRNA and homology arms (+cargo). The sgRNA was designed using Benchling’s CRISPR algorithm (https://benchling.com/) set to C. difficile 630 genome, with an NGG PAM targeting genes ( ptp, ptc, tcf, tfp, hyp ) for deletion or sgRN-BM6 55 for complementation/exchange mutants. The resulting sgRNA was digested with AscI and AatII.…”

Section: Methodsmentioning

confidence: 99%

A tale of two phage tails: Engineering the host range of bacteriophages infectingClostridioides difficile

Steczynska,

Kerr,

Kelly

et al. 2023

Preprint

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Clostridioides difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea across the globe. Although treatable with a restricted number of antibiotics, the emergence of resistant variants and high relapse rates necessitate alternative countermeasures. Phage therapy represents an attractive option. However, its implementation is handicapped by the narrow host specificity of the C. difficile bacteriophages isolated to date. One strategy to rationally expand phage host range would be to make appropriate modifications to the phage receptor binding protein (RBP). Here, we identify the tail fibre as the RBP of two Myoviridae phages, ΦCD1801 and ΦCD2301, which were previously isolated and propagated using the C. difficile strains CD1801 (RT078) and CD2301 (RT014), respectively. Contrary to studies into reprogramming the host ranges of phage of other bacterial other species, exchanging the tail fibre genes (tcf/tfp) alone between the two phage was insufficient to change host specificity. Rather, alterations to host range were dependent their exchange together with a putative chaperone encoded by hyp, localised adjacent to the tail fibre gene. Capitalising on this discovery, CRISPR/Cas9 was used to change the host range of one phage to that of the other by swapping the respective tcf/tfp and hyp genes. Significantly, one of the resulting mutants, surpassed both parental phages in terms of host range and efficiency of infection. This is the first time that genome engineering has successfully expanded the host range of a C. difficile phage, a prerequisite for implementing phage for the treatment of CDI.

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Section: Methodsmentioning

confidence: 99%

A tale of two phage tails: Engineering the host range of bacteriophages infectingClostridioides difficile

Steczynska,

Kerr,

Kelly

et al. 2023

Preprint

Self Cite

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“…However, the time gained during design and assembly can be quickly lost again if mixed colonies need to be restreaked (especially for slow-growing organisms like C. autoethanogenum). Initial screening of mutants is also more difficult than with standard homology-directed knockouts (Seys et al, 2020), as a colony PCR cannot readily identify the desired SNPs through simple gel electrophoresis.…”

Section: Locusmentioning

confidence: 99%

Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations

Seys,

Humphreys,

Tomi-Andrino

et al. 2023

Front. Bioeng. Biotechnol.

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Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes that lack effective DSB repair pathways such as homologous recombination (HR) or non-homologous end-joining (NHEJ). However, few applications of base editors have been reported in prokaryotes to date, and their advantages and drawbacks have not been systematically reported. Here, we used the base editors Target-AID and Target-AID-NG to introduce nonsense mutations into four different coding sequences of the industrially relevant Gram-positive bacterium Clostridium autoethanogenum. While up to two loci could be edited simultaneously using a variety of multiplexing strategies, most colonies exhibited mixed genotypes and most available protospacers led to undesired mutations within the targeted editing window. Additionally, fifteen off-target mutations were detected by sequencing the genome of the resulting strain, among them seven single-nucleotide polymorphisms (SNP) in or near loci bearing some similarity with the targeted protospacers, one 15 nt duplication, and one 12 kb deletion which removed uracil DNA glycosylase (UDG), a key DNA repair enzyme thought to be an obstacle to base editing mutagenesis. A strategy to process prokaryotic single-guide RNA arrays by exploiting tRNA maturation mechanisms is also illustrated.

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An adapted method for Cas9-mediated editing reveals the species-specific role of β-glucoside utilization driving competition betweenKlebsiellaspecies

d. H. Almási,

Knischewski,

Osbelt

et al. 2023

Preprint

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Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of theKlebsiella oxytocaspecies complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition againstKlebsiella pneumoniae. The system allowed efficient and precise editing via enhanced lambda Red expression and functionally-validated complementation with the use of universal ‘bookmark’ targets inK. oxytoca, Klebsiella michiganensis, andKlebsiella grimontii. We revealed that the carbohydrate permease CasA is critical inex vivoassays forK. pneumoniaeinhibition byK. oxytocabut is neither sufficient nor required forK. michiganensisandK. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition.* ImportanceCas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion inKlebsiella pneumoniaeand demonstrate after optimization its utility for gene editing in three members of theKlebsiella oxytocaspecies complex (KoSC) namelyK. oxytoca, K. michiganensisandK. grimontii. We then adapted a recently developed protocol for functional complementation based on universal ‘bookmark’ targets applicable to all tested species. In summary, species specific adaptation of state-of-the-art genetic tools allow efficient gene deletion and complementation in types strains as well as natural isolates of KoSC members to study microbial interactions.

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A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

Cited by 4 publications

References 17 publications

A tale of two phage tails: Engineering the host range of bacteriophages infectingClostridioides difficile

A tale of two phage tails: Engineering the host range of bacteriophages infectingClostridioides difficile

Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations

An adapted method for Cas9-mediated editing reveals the species-specific role of β-glucoside utilization driving competition betweenKlebsiellaspecies

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