New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.     

Random segment deletion based on IS<Emphasis Type="Italic">31831</Emphasis> and Cre/<Emphasis Type="Italic">loxP</Emphasis> excision system in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

A simple and random genome deletion method combining insertion sequence (IS) element IS31831 and the Cre/loxP excision system generated 42 Corynebacterium glutamicum mutants (0.2–186 kb). A total of 393.6 kb (11.9% of C. glutamicum R genome) coding for 331 genes was confirmed to be nonessential under standard laboratory conditions. The deletion strains, generated using only two vectors, varied not only in their lengths but also the location of the deletion along the C. glutamicum R genome. By comparing and analyzing the generated deletion strains, identification of nonessential genes, the roles of genes of hitherto unknown function, and gene–gene interactions can be easily and efficiently determined. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Multiple large segment deletion method for Corynebacterium glutamicum

A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering.     

Large-Scale Engineering of the Corynebacterium glutamicum Genome

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.     

New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.     

Deletion of cgR_1596 and cgR_2070, encoding NlpC/P60 proteins, causes a defect in cell separation in Corynebacterium glutamicum R

In previous work, random genome deletion mutants of Corynebacterium glutamicum R were generated using the insertion sequence (IS) element IS31831 and the Cre/loxP excision system. One of these mutants, C. glutamicum strain RD41, resulting from the deletion of a 10.1-kb genomic region (ΔcgR_1595 through cgR_1604) from the WT strain, showed cell elongation, and several lines appeared on the cell surface (bamboo shape). The morphological changes were suppressed by overexpression of cgR_1596. Single disruption of cgR_1596 in WT C. glutamicum R resulted in morphological changes similar to those observed in the RD41 strain. CgR_1596 has a predicted secretion signal peptide in the amino-terminal region and a predicted NlpC/P60 domain, which is conserved in cell wall hydrolases, in the carboxyl-terminal region. In C. glutamicum R, CgR_0802, CgR_1596, CgR_2069, and CgR_2070 have the NlpC/P60 domain; however, only simultaneous disruption of cgR_1596 and cgR_2070, and not cgR_2070 single disruption, resulted in cell growth delay and more severe morphological changes than disruption of cgR_1596. Transmission electron microscopy revealed multiple septa within individual cells of cgR_1596 single and cgR_1596-cgR_2070 double disruptants. Taken together, these results suggest that cgR_1596 and cgR_2070 are involved in cell separation and cell growth in C. glutamicum.     

Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71

The Cre/loxP recombination system is a commonly used tool to alter the mouse genome in a conditional manner by deletion or inversion of loxP-flanked DNA segments. While Cre-mediated deletion is essentially unidirectional, inversion is reversible and therefore does not allow the stable alteration of gene function in cells that constitutively express Cre. Site-directed mutagenesis yielded a pair of asymmetric loxP sites (lox66 and lox71) that display a favorable forward reaction equilibrium. Here, we demonstrate that lox66/lox71 mediates efficient and predominantly unidirectional inversion of a switch substrate targeted to the mouse genome in combination with either inducible or cell type-specific cre-transgenes in vivo.     

Site-directed integration system using a combination of mutant <Emphasis Type="Italic">lox</Emphasis> sites for <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an optimal C. glutamicum genome, a precise site-directed gene integration method was developed by using a pair of mutant lox sites, one a right element (RE) mutant lox site and the other a left element (LE) mutant lox site. Two DNA fragments, 5.7 and 10.2 kb-long, were successfully integrated into the genome. The recombination efficiency of this system compared to that obtained by single crossover by homologous recombination was 2 orders of magnitude higher. Moreover, the integrated DNA remained stably maintained on removal of Cre recombinase. The Cre/mutant lox system thus represents a potentially attractive tool for integration of foreign DNA in the course of the engineering of C. glutamicum traits.     

An accumulative site‐specific gene integration system using cre recombinase‐mediated cassette exchange

The Cre‐loxP system is frequently used for site‐specific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site‐specific gene integration system using Cre recombinase and mutated loxPs in which the Cre‐mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms. Biotechnol. Bioeng. 2010;105: 1106–1114. © 2009 Wiley Periodicals, Inc.     

Cre-loxP Recombination System for Large Genome Rearrangements in Lactococcus lactis

We have used a new genetic strategy based on the Cre-loxP recombination system to generate large chromosomal rearrangements in Lactococcus lactis. Two loxP sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal loxP sites was highly efficient (approximately 1 × 10⁻¹/cell) when the Cre recombinase was provided in trans, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-loxP recombination system described can potentially be used for other gram-positive bacteria without further modification.     

Reactivation of an integrated disabled viral vector using a Cre-loxP recombination system in Arabidopsis thaliana

Summary We developed an inactivated DNA replicon of Turnip Mosaic Virus (TuMV), which was reactivated by a recombination event based on the Cre-loxP system. Viral replication was prevented by the insertion of a translation terminator sequence flanked by two loxP sites at the junction of the P1–HCPro-coding genes. In vitro recombination was tested with purified Cre, which excised the floxed sequence from the TuMV DNA, leaving a single loxP site in the reactivated viral genome, and restored the open reading frame of the replicon. Arabidopsis thaliana plants were made transgenic for the inactivated TuMV replicon. Removal of the translation terminator sequence was achieved by the controlled expression of Cre. Delivery of the Cre recombinase to the transgenic plants was obtained by three methods: agroinfiltration, PVX-based production, or transgenic chemical-inducible expression. In each case, reactivation of TuMV replication was observed.     

Expression of Cre recombinase during transient phage infection permits efficient marker removal in Streptomyces

We report a system for the efficient removal of a marker flanked by two loxP sites in Streptomyces coelicolor, using a derivative of the temperate phage C31 that expresses Cre recombinase during a transient infection. As the test case for this recombinant phage (called Cre-phage), we present the construction of an in-frame deletion of a gene, pglW, required for phage growth limitation or Pgl in S.coelicolor. Cre-phage was also used for marker deletion in other strains of S.coelicolor.     

An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum

Corynebacterium glutamicum is an important industrial microorganism, but the availability of tools for its genetic modification has lagged compared to other model microorganisms such as Escherichia coli. Despite great progress in CRISPR-based technologies, the most feasible genome editing method in C. glutamicum is suicide plasmid-mediated, the editing efficiency of which is low due to high false-positive rates of sacB counter selection, and the requirement for tedious two-round selection and verification of rare double-cross-over events. In this study, an rpsL mutant conferring streptomycin resistance was harnessed for counter selection, significantly increasing the positive selection rate. More importantly, with the aid of high selection efficiencies through the use of antibiotics, namely kanamycin and streptomycin, the two-step verification strategy can be simplified to just one-step verification of the final edited strain. As proof of concept, a 2.5-kb DNA fragment comprising aroG^fbrpheA^fbr expressing cassettes was integrated into the genome of C. glutamicum, with an efficiency of 20% out of the theoretical 50%. The resulting strain produced 110 mg l⁻¹ l -tyrosine in shake-flask fermentation. This updated suicide plasmid-mediated genome editing system will greatly facilitate genetic manipulations including single nucleotide mutation, gene deletion and gene insertion in C. glutamicum and can be easily applied to other microbes.     

A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites

A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.     

Site-directed recombination in the genome of transgenic tobacco

Summary The plant genome responds to the bacteriophage P1-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase 11 gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%–100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2–3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing site-specific integration and regulation of gene expression.     

Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs. We replaced a part of the T-cell receptor γ (TCR Vγ) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique. The resulting ES cell clones containing the mutant allele (Vγ^LNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild-type female mice. One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted Vγ locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (Vγ^Δ) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the Vγ^Δ locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined Vγ^LNL locus. Thus, the frequency of Cre-mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS-Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the Vγ^LNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes. Mol Reprod Dev 46:109–113, 1997. © 1997 Wiley-Liss, Inc.     

A novel reporter rat strain that expresses LacZ upon Cre‐mediated recombination

The recent widespread application of Cre/loxP technology has resulted in a new generation of conditional animal models that can better recapitulate many salient features of human disease. These models benefit from the ability to monitor the expression and functionality of Cre protein. We have generated a conditional (Cre/loxP dependent) LacZ reporter rat (termed the LacZ541 rat) to monitor Cre in transgenic rats. When LacZ541 rats were bred with another transgenic rat line expressing Cre recombinase under the control of the CAG promoter, LacZ/Cre double transgenic embryos displayed ubiquitous expression of LacZ, and when LacZ541 rats were bred with transgenic rats expressing Cre/loxP‐dependent oncogenic H‐ or K‐ras, LacZ was expressed in the lesions resulting from the activation of the oncogene. The LacZ541 rat enables evaluation of the performance of Cre‐expressing systems which are based upon transgenic rats or somatic gene transfer vectors and provides efficient and simple lineage marking. genesis 51:268–274. © 2013 Wiley Periodicals, Inc.     

Engineering Corynebacterium glutamicum with a comprehensive genomic library and phage-based vectors

The Gram-positive bacterium Corynebacterium glutamicum sustains the industrial production of chiral molecules such as L-amino acids. Through heterologous gene expression, C. glutamicum is becoming a sustainable source of small organic molecules and added-value chemicals. The current methods to implement heterologous genes in C. glutamicum rely on replicative vectors requiring lasting selection or chromosomal integration using homologous recombination. Here, we present a set of dedicated and transversal tools for genome editing and gene delivery into C. glutamicum. We generated a cosmid-based library suitable for efficient double allelic exchange, covering more than 94% of the chromosome with an average 5.1x coverage. We employed the library and an iterative marker excision system to generate the carotenoid-free C. glutamicum BT1-C31-Albino (BCA) host, featuring the attachment sites for actinophages ϕC31 and ϕBT1 for one-step chromosomal integration. As a proof-of-principle, we employed a ϕC31-based integration and a Cre system for the markerless expression of the type III polyketide synthase RppA, and a ϕBT1-based integration system for the expression of the phosphopantetheinylation-dependent non-ribosomal peptide synthetase BpsA in the C. glutamicum BCA host. The developed genomic library and microbial host, and the characterized molecular tools will contribute to the study of the physiology and the rise of C. glutamicum as a leading host for drug discovery.     

Site-specific integration of transgene targeting an endogenous <Emphasis Type="Italic">lox-</Emphasis>like site in early mouse embryos

Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (ψlox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/ψlox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endogenous lox-like sequence termed pseudo-loxm5 (ψloxm5) in early mouse embryos. We characterized the Cre/ψloxm5 system in embryonic environment. Cre-expressing plasmid and a transgene (CMV/LacZ gene) flanked by ψloxm5 and ψloxcorem5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers, and the recombination products were investigated. The results show that the ψloxm5 site is an active substrate for Cre-mediated recombination in the mouse embryonic environment. The transgenesis efficiency was up to 27% (6/22). The site-specific integration of the transgene into the endogenous ψloxm5 site was found in 50 % of the transgenic pups. Our findings demonstrated that the Cre/ψloxm5 integrative recombination system is an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.