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

Jiang, Yu; Chen, Biao; Duan, Chunlan; Sun, Bingbing; Yang, Junjie; Yang, Sheng

doi:10.1128/aem.04023-14

Cited by 1,050 publications

(1,076 citation statements)

References 54 publications

(60 reference statements)

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“…5). In addition, our approach is easily amendable to multiplexing through the targeting of multiple chromosomal loci (27) and circumvents the use of chromosomal antibiotic markers and Flp-mediated excision for antibiotic marker recycling. Hence, the resulting mutant genome is both markerless and "scar"-less, as no FRT "scar" sites remain following recombination.…”

Section: Discussionmentioning

confidence: 99%

“…In efforts demanding both large chromosome deletion and large heterologous DNA insertion, we recommend utilizing traditional, yet more laborious, lambda Red protocols in the absence of CRISPR/ Cas9 selection (3, 10), as rare recombination events can be selected directly via chromosomal antibiotic resistance. Alternatively, increasing the size of homology arms beyond the common 40-nt limit utilized in this study has been shown to enhance recombination (27), which could enable detection of low-efficiency events. It has also been reported that homologous recombination varies drastically between genomic loci (40), presumably due to secondary-structure and sequence-dependent effects.…”

Section: Discussionmentioning

confidence: 99%

“…Using the CRISPR/Cas9 system for selection of E. coli genome editing, up to 65% of cells were found to possess the desired chromosomal mutation specified by the synthetic oligonucleotide (24), rendering CRISPR/Cas9 recombineering the most powerful methodology for engineering bacterial genomes to date (26). In addition to E. coli and Streptococcus (24,27), this technology has recently been adapted for use with lactic acid bacteria (28), Streptomyces (29), and Clostridium (30).…”

mentioning

confidence: 99%

“…We performed preliminary gene replacements to demonstrate the utility of this approach, extensively probed its capacity for generating large chromosomal deletions and insertions, and examined its advantages over traditional recombineering approaches in the absence of CRISPR/Cas9 selection (3,10). This method overcomes the dependence on flippase (Flp) recombination, avoids the creation of chromosomal "scar" sites (i.e., Flp recognition target [FRT] scar sites), is adaptable to multiplexing (27), and can be performed in a shorter period than previous protocols. Accordingly, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 recombineering simplifies the construction of markerless gene replacement mutants of E. coli and serves as an effective tool for strain optimization, metabolic engineering, and synthetic biology.…”

mentioning

confidence: 99%

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Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

Pyne

Moo‐Young

Chung

et al. 2015

Appl Environ Microbiol

211

188

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cTo date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and "scar"-less. Since its inception in 1998, phage recombinase-mediated homologous recombination, also known as recombineering, has revolutionized bacterial genetics, synthetic biology, and metabolic engineering (1, 2). Relying on either RecET from the Rac prophage (2) or the bacteriophage lambda Red proteins, Exo, Beta, and Gam (1), recombineering allows simple and efficient construction of gene knockout mutants via homologous recombination of a double-stranded DNA (dsDNA) PCR product with bacterial chromosomes (3). This work has led to the construction of an Escherichia coli K-12 single-gene knockout library (the Keio collection [4]), enabled widespread use of chromosome-encoded expression platforms for metabolic and strain engineering (5-11), and simplified conventional recombinant DNA technology through the advent of ligation-independent DNA cloning (2, 12). Automated, high-throughput, and multiplexed genome editing applications have also been envisioned, including multiplexed automated genome engineering (MAGE), generating up to 4.3 billion genomic mutants per day (13). Finally, phage-mediated recombineering systems have been identified and exploited for use in an array of bacterial genera (14-19).Recombineering can be carried out using either singlestranded DNA (ssDNA) or dsDNA substrates. Since recombineering occurs through an entirely ssDNA intermediate (20), ssDNA recombineering requires only an ssDNA recombinase (RecT or Beta) for recombination (21), whereas recombination of dsDNA substrates requires both an exonuclease (RecE or Exo) and its associated recombinase (RecT or Beta). For this reason, recombination of ssDNA substrates, such as oligonucleotides, is simpler, more efficient, and better understood ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

Pyne

Moo‐Young

Chung

et al. 2015

Appl Environ Microbiol

211

188

View full text Add to dashboard Cite

show abstract

“…(2) Many p rotospacer adjacent motif (PAM) sites may lead to undesired cleavage of DNA regions [101]; to resolve this problem, bioinformatics tools are being developed at whole genome sequence level to improve specificity [102]. (3) Codon usage varies across species and may affect Cas9 translation; several codon-optimized versions of Cas9 genes have therefore been harnessed for several individual crops [102] and there may be need for a cassava codon optimized Cas9.…”

Section: Clustered Regularly Interspaced Short Palindromic Repeat (Crmentioning

confidence: 99%

Recent Biotechnological Advances in the Improvement of Cassava

Fondong¹,

Rey²

2018

Cassava

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Cassava (Manihot esculenta Crantz) is the fourth most important source of carbohydrates for human consumption in the tropics and thus occupies a uniquely important position as a food security crop for smallholder farmers. Consequently, cassava improvement is of high priority to most national agricultural research institutions in the tropics. With advances in functional genomics and genome editing approaches in this post genomics era, there are unprecedented opportunities and potential to accelerate the improvement of this important crop. These new technologies will need to be directed toward addressing major cassava production constraints, notably virus resistance, protein content, tolerance to drought and reduction of hydrogen cyanide content. Here, we discuss the important role novel functional genomics and genome editing technologies have and will continue to play in cassava improvement efforts. These approaches, including artificial miRNA (amiRNA), trans-acting small interfering RNA (tasiRNA), clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9), and Targeting Induced Local Lesions IN Genomes (TILLING), have been shown to be effective in addressing major crop production constraints. In addition to reviewing specific applications of these technologies in cassava improvement, this chapter discusses specific examples being deployed in the amelioration of cassava or of other crops that could be applied to cassava in future.

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Cytochrome bd‐I from Escherichia coli is catalytically active in the absence of the CydH subunit

et al. 2022

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Cytochrome bd-I from Escherichia coli is a terminal oxidase in the respiratory chain that plays an important role under stress conditions. Cytochrome bd-I was thought to consist of the major subunits CydA and CydB plus the small CydX subunit. Recent high-resolution structures of cytochrome bd-I demonstrated the presence of an additional subunit, CydH/CydY (called CydH here), the function of which is unclear. In this report, we show that in the absence of CydH, cytochrome bd-I is catalytically active, can sustain bacterial growth and displays haem spectra and susceptibility for haem-binding inhibitors comparable to the wild-type enzyme. Removal of CydH did not elicit catalase activity of cytochrome bd-I in our experimental system. Taken together, in the absence of the CydH subunit cytochrome bd-I retained key enzymatic properties.

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

Cited by 1,050 publications

References 54 publications

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

Recent Biotechnological Advances in the Improvement of Cassava

Cytochrome bd‐I from Escherichia coli is catalytically active in the absence of the CydH subunit

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