High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

Yang, Junjie; Sun, Bingbing; Huang, He; Jiang, Yu; Diao, Liuyang; Chen, Biao; Xu, Chongmao; Wang, Xin; Liu, Jinle; Jiang, Weihong; Yang, Sheng

doi:10.1128/aem.00313-14

Cited by 80 publications

(64 citation statements)

References 39 publications

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“…Compared to published scarless genome modification methods, such as those involving sacB (12), I-SceI (12,19,20), and MAGE (22,23), the CRISPRbased targeted genome modification method can perform multiple gene insertions or deletions, whereas sacB or I-SceI could be used to modify only single targets each time. ssDNA oligonucleotide-mediated MAGE was used successfully for multiple allelic exchange, but small-fragment (30 bp) insertion decreased mutation efficiency dramatically (12,22).…”

Section: Discussionmentioning

confidence: 99%

“…The efficiency of introduction of mutations mediated by homologous recombination can be improved (i) by using counterselection markers, such as the typical sacB-based method (12), and (ii) by improving the frequency of homologous recombination by using phage-derived recombinases (RecET and -Red) (13)(14)(15), applying doublestranded (16,17) or single-stranded donor DNAs (18), or inducing double-stranded breaks (DSBs) in a chromosomal target using I-SceI (12,19,20). The -Red recombinase method (13) and group II intron retrotransposition (21) leave scars in the genome that limit their application in allelic exchange.…”

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confidence: 99%

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Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System

Jiang

Chen

Duan

et al. 2015

Appl Environ Microbiol

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An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System

Jiang

Chen

Duan

et al. 2015

Appl Environ Microbiol

Self Cite

1,047

1,003

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“…For instance, I‐SceI is an endonuclease recognizing an 18 bp sequence and was exploited to generate DSB on the E. coli chromosome and plasmid for gene integration. In combination with λ‐Red proteins, this method was successful to integrate a DNA fragment up to 7 kb (Kuhlman and Cox, ; Yang et al, ) and could mediate gene integration into multiple loci (Yang et al, ). However, due to the lack of natural I‐SceI recognition site in E. coli , pre‐integration of a landing pad encompassing the recognition sequence is necessary, thus entailing two rounds of homologous recombination and selection processes.…”

Section: Discussionmentioning

confidence: 99%

Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9

Chung

I-Hsin

Sung

et al. 2016

Biotech & Bioengineering

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Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc.

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“…Pioneered by Murphy [20] and later modified by Datsenko and Wanner [21], this method involves the introduction of single- or double-stranded DNA with chromosomal homology regions for recombination [22]. Since its conception, this editing strategy has been made more efficient by modifications to the method developed by Wanner [23–25]. This has also been readily adapted to pathogenic bacterial strains for investigating the roles of genes in pathogenesis [23, 26–29].…”

Section: Main Textmentioning

confidence: 99%

Bacterial genome engineering and synthetic biology: combating pathogens

et al. 2016

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BackgroundThe emergence and prevalence of multidrug resistant (MDR) pathogenic bacteria poses a serious threat to human and animal health globally. Nosocomial infections and common ailments such as pneumonia, wound, urinary tract, and bloodstream infections are becoming more challenging to treat due to the rapid spread of MDR pathogenic bacteria. According to recent reports by the World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC), there is an unprecedented increase in the occurrence of MDR infections worldwide. The rise in these infections has generated an economic strain worldwide, prompting the WHO to endorse a global action plan to improve awareness and understanding of antimicrobial resistance. This health crisis necessitates an immediate action to target the underlying mechanisms of drug resistance in bacteria.ResearchThe advent of new bacterial genome engineering and synthetic biology (SB) tools is providing promising diagnostic and treatment plans to monitor and treat widespread recalcitrant bacterial infections. Key advances in genetic engineering approaches can successfully aid in targeting and editing pathogenic bacterial genomes for understanding and mitigating drug resistance mechanisms. In this review, we discuss the application of specific genome engineering and SB methods such as recombineering, clustered regularly interspaced short palindromic repeats (CRISPR), and bacterial cell-cell signaling mechanisms for pathogen targeting. The utility of these tools in developing antibacterial strategies such as novel antibiotic production, phage therapy, diagnostics and vaccine production to name a few, are also highlighted.ConclusionsThe prevalent use of antibiotics and the spread of MDR bacteria raise the prospect of a post-antibiotic era, which underscores the need for developing novel therapeutics to target MDR pathogens. The development of enabling SB technologies offers promising solutions to deliver safe and effective antibacterial therapies.

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High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

Abstract: Genetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of the Escherichia coli chromosome, a two-plasmid method involving lambda Red (

Cited by 80 publications

References 39 publications

Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System

Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System

Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9

Bacterial genome engineering and synthetic biology: combating pathogens

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