Multiplex genome editing by natural transformation

Dalia, Ankur B.; McDonough, EmilyKate; Camilli, Andrew

doi:10.1073/pnas.1406478111

Cited by 212 publications

(266 citation statements)

References 35 publications

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“…Furthermore, for inactivation of the MMR system, the host has to be modified beforehand, greatly limiting the ease of use and narrowing the applicable range of organisms. Although a method expanding multiplex genome editing has recently been described [MuGENT (37)], it is applicable only to naturally transformable bacteria (for other potential limitations, see Table S1). Building on prior works, our study addresses the above mentioned three major problems-ease of use, off-target mutagenesis, and portability across species-in a single framework.…”

Section: Discussionmentioning

confidence: 99%

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

Nyerges

Csörgő

Nagy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

207

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Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.genome engineering | synthetic biology | recombineering | off-target effects | methyl-directed mismatch repair

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Section: Discussionmentioning

confidence: 99%

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

Nyerges

Csörgő

Nagy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

207

256

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“…However, ADP1 has many potential advantages as a versatile host microorganism for synthetic biology (6)(7)(8), especially considering recent studies describing multiplex genome editing in naturally transformable organisms (52). The genetic stability of ADP1 could be improved by domesticating its genome by deleting IS elements, cryptic prophages, potential toxin-antitoxin cassettes, and other uncharacterized genes that have accumulated during its prior existence in the wild outside the laboratory, as has been done in the "clean-genome" E. coli MDS42 strain (3).…”

Section: Discussionmentioning

confidence: 99%

Genome Instability Mediates the Loss of Key Traits by Acinetobacter baylyi ADP1 during Laboratory Evolution

et al. 2015

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Acinetobacter baylyi ADP1 has the potential to be a versatile bacterial host for synthetic biology because it is naturally transformable. To examine the genetic reliability of this desirable trait and to understand the potential stability of other engineered capabilities, we propagated ADP1 for 1,000 generations of growth in rich nutrient broth and analyzed the genetic changes that evolved by whole-genome sequencing. Substantially reduced transformability and increased cellular aggregation evolved during the experiment. New insertions of IS1236 transposable elements and IS1236-mediated deletions led to these phenotypes in most cases and were common overall among the selected mutations. We also observed a 49-kb deletion of a prophage region that removed an integration site, which has been used for genome engineering, from every evolved genome. The comparatively low rates of these three classes of mutations in lineages that were propagated with reduced selection for 7,500 generations indicate that they increase ADP1 fitness under common laboratory growth conditions. Our results suggest that eliminating transposable elements and other genetic failure modes that affect key organismal traits is essential for improving the reliability of metabolic engineering and genome editing in undomesticated microbial hosts, such as Acinetobacter baylyi ADP1.T he large-scale engineering of genome sequences is a growing area of synthetic biology and bioengineering with many applications (1, 2). Projects such as the development of reduced bacterial genomes (3), modifying the genetic code (4), and optimizing metabolic pathways (5) require whole-scale genetic rewriting of genomes in order to accommodate progressively more ambitious goals. The process of making such modifications is a resourceintensive undertaking with significant technical, human-resource, and time costs. Acinetobacter baylyi ADP1 has been proposed as a model organism for metabolic engineering, synthetic biology, and genome evolution studies because it is naturally transformable (6-8). During normal growth in the laboratory, ADP1 expresses competence machinery that enables it to efficiently import extracellular DNA without any sequence constraints (9, 10). By designing DNA constructs with homology to the ADP1 genome, targeted deletions or insertions of new genes can be made with high efficiency without the need for artificial transformation techniques or the expression of heterologous genes, as is typically required when engineering the Escherichia coli genome (5, 11). Thus, ADP1 could be a useful chassis organism for studies that require large-scale or combinatorial genome modifications.One known challenge associated with ADP1, however, is that the competence phenotype is unstable when it is cultured in the laboratory (12). In general, genetic reliability is a key trait sought after in chassis organisms; one does not want to design a DNA sequence only to see it be rapidly deleted or mutated when placed in a living organism. In this vein, efforts to engineer E. coli v...

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“…The desired incorporation of the entire classical CRISPR-Cas system in E7946 was confirmed by PCR; in the case of the parent strain used in our plasmid-based CRISPR assays, the expected sequence of the incorporated CRISPR-Cas system was confirmed by whole-genome sequencing using genomic libraries generated as described previously (30) and sequenced using an Illumina HiSeq 2000 sequencing system. The kanamycin resistance cassette was removed using cotransformation (34) of the wild-type locus with a selected product which replaced lacZ with an Spc resistance (Spc r ) marker (generating strain KS802). The overexpression construct for P tac -CRISPR-cas was generated by SOE PCR using a fragment derived from a kanamycin-resis-tant derivative of a Tn10 plasmid (33) and was introduced via natural transformation to generate strain KS916.…”

Section: Methodsmentioning

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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Multiplex genome editing by natural transformation

Cited by 212 publications

References 35 publications

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

Genome Instability Mediates the Loss of Key Traits by Acinetobacter baylyi ADP1 during Laboratory Evolution

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

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