Characterisation of a new reporter system allowing high throughput in planta screening for recombination events before and after controlled DNA double strand break induction

DNA double strand breaks (DSBs) are created either by DNA damaging reagents or in a programmed manner, for example during meiosis. Homologous recombination (HR) can be used to repair DSBs, a process vital both for cell survival and for genetic rearrangement during meiosis. In order to easily quantify this mechanism, a new HR reporter gene that is suitable for the detection of rare recombination events in high-throughput screens was developed in Arabidopsis thaliana. This reporter, pPNP, is composed of two mutated Pat genes and has also one restriction site for the meganuclease I-SceI. A functional Pat gene can be reconstituted by an HR event giving plants which are resistant to the herbicide glufosinate. The basal frequency of intra-chromosomal recombination is very low (10^?5) and can be strongly increased by the expression of I-SceI which creates a DSB. Expression of I-SceI under the control of the 35S CaMV promoter dramatically increases HR frequency (10,000 fold); however the measured recombinant events are in majority somatic. In contrast only germinal recombination events were measured when the meganuclease was expressed from a floral-specific promoter. Finally, the reporter was used to test a dexamethasone inducible I-SceI which could produce up to 200× more HR events after induction. This novel inducible I-SceI should be useful in fundamental studies of the mechanism of repair of DSBs and for biotechnological applications.     

I-SceI-mediated plasmid deletion and intra-molecular recombination in Spiroplasma citri

S. citri wild-type strain GII3 carries six plasmids (pSci1 to -6) that are thought to encode determinants involved in the transmission of the spiroplasma by its leafhopper vector. In this study we report the use of meganuclease I-SceI for plasmid deletion in S. citri. Plasmids pSci1NT-I and pSci6PT-I, pSci1 and pSci6 derivatives that contain the tetM selection marker and a unique I-SceI recognition site were first introduced into S. citri strains 44 (having no plasmid) and GII3 (carrying pSci1-6), respectively. Due to incompatibility of homologous replication regions, propagation of the S. citri GII3 transformant in selective medium resulted in the replacement of the natural pSci6 by pSci6PT-I. The spiroplasmal transformants were further transformed by an oriC plasmid carrying the I-SceI gene under the control of the spiralin gene promoter. In the S. citri 44 transformant, expression of I-SceI resulted in rapid loss of pSciNT-I showing that expression of I-SceI can be used as a counter-selection tool in spiroplasmas. In the case of the S. citri GII3 transformant carrying pSci6PT-I, expression of I-SceI resulted in the deletion of plasmid fragments comprising the I-SceI site and the tetM marker. Delineating the I-SceI generated deletions proved they had occurred though recombination between homologous sequences. To our knowledge this is the first report of I-SceI mediated intra-molecular recombination in mollicutes.     

New ultrarare restriction site-carrying transposons for bacterial genomics

Electrophoretic separation of macrorestriction fragments containing a particular genomic interval has until recently depended on fortuitously placed native rare restriction sites. We present new IS10-based transposons carrying the yeast intron-encoded I-SceI restriction site which is absent from most prokaryotic and eukaryotic genomes. Construction of the plasmid vectors containing them is described. Analysis by conventional or Pulsed Field gel electrophoresis of the DNA fragments generated by the I-SceI digestion reveals the physical distance between genomic insertions of these transposons: use of the same approach to subdivide the chromosome of Escherichia coli K-12 into equivalently sized contiguous/nonoverlapping I-SceI fragments is demonstrated. Because coordinates for the loci delimited by their insertions can be readily determined in different isolates by either physical or genetic manipulations, these transposons allow sufficient flexibility for species-wide bacterial genomics.     

Analysis of Gene Targeting and Intrachromosomal Homologous Recombination Stimulated by Genomic Double-Strand Breaks in Mouse Embryonic Stem Cells

To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.     

Targeted mutagenesis in the progeny of maize transgenic plants

We have demonstrated that targeted mutagenesis can be accomplished in maize plants by excision, activation, and subsequent elimination of an endonuclease in the progeny of genetic crosses. The yeast FLP/FRT site-specific recombination system was used to excise and transiently activate the previously integrated yeast I-SceI homing endonuclease in maize zygotes and/or developing embryos. An artificial I-SceI recognition sequence integrated into genomic DNA was analyzed for mutations to indicate the I-SceI endonuclease activity. Targeted mutagenesis of the I-SceI site occurred in about 1% of analyzed F1 plants. Short deletions centered on the I-SceI-produced double-strand break were the predominant genetic lesions observed in the F1 plants. The I-SceI expression cassette was not detected in the mutant F1 plants and their progeny. However, the original mutations were faithfully transmitted to the next generation indicating that the mutations occurred early during the F1 plant development. The procedure offers simultaneous production of double-strand breaks and delivery of DNA template combined with a large number of progeny plants for future gene targeting experiments.     

I-SceI endonuclease: a new tool for DNA repair studies and genetic manipulations in streptomycetes

Actinomycetes are Gram-positive bacteria with a complex life cycle. They produce many pharmaceutically relevant secondary metabolites, including antibiotics and anticancer drugs. However, there is a limited number of biotechnological applications available as opposed to genetic model organisms like Bacillus subtilis or Escherichia coli. We report here a system for the functional expression of a synthetic gene encoding the I-SceI homing endonuclease in several streptomycetes. Using the synthetic sce(a) gene, we were able to create controlled genomic DNA double-strand breaks. A mutagenesis system, based on the homing endonuclease I-SceI, has been developed to construct targeted, non-polar, unmarked gene mutations in Streptomyces sp. Tü6071. In addition, we have shown that homologous recombination is a major pathway in streptomycetes to repair an I-SceI-generated DNA double-strand break. This novel I-SceI-based tool will be useful in fundamental studies on the repair mechanism of DNA double-strand breaks and for a variety of biotechnological applications.     

Improvements to a Markerless Allelic Exchange System for Bacillus anthracis

A system was previously developed for conducting I-SceI-mediated allelic exchange in Bacillus anthracis. In this system, recombinational loss of a chromosomally-integrated allelic exchange vector is stimulated by creation of a double-stranded break within the vector by the homing endonuclease I-SceI. Although this system is reasonably efficient and represents an improvement in the tools available for allelic exchange in B. anthracis, researchers are nonetheless required to “pick and patch” colonies in order to identify candidate "exchangeants." In the present study, a number of improvements have been made to this system: 1) an improved I-SceI-producing plasmid includes oriT so that both plasmids can now be introduced by conjugation, thus avoiding the need for preparing electro-competent cells of each integration intermediate; 2) antibiotic markers have been changed to allow the use of the system in select agent strains; and 3) both plasmids have been marked with fluorescent proteins, allowing the visualization of plasmid segregation on a plate and obviating the need for “picking and patching.” These modifications have made the process easier, faster, and more efficient, allowing for parallel construction of larger numbers of mutant strains. Using this improved system, the genes encoding the tripartite anthrax toxin were deleted singly and in combination from plasmid pXO1 of Sterne strain 34F2. In the course of this study, we determined that DNA transfer to B. anthracis could be accomplished by conjugation directly from a methylation-competent E. coli strain.     

Efficient repair of DNA double-strand breaks in malignant cells with structural instability

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1α) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1α promoter from the TK gene, or deletion of either the EF1α promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.     

Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we stably transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. Cells were then electroporated with a plasmid expressing endonuclease I-SceI to induce a DSB, and clones that had lost tk function were selected. In a previous study of DSB-induced tk-deficient clones, we found that ~8% of recovered tk mutations involved the capture of one or more DNA fragments at the DSB site. Almost half of the DNA capture events involved the I-SceI expression plasmid, and several events involved retrotransposable elements. To learn whether only certain DNA sequences or motifs are efficiently captured, in the current work we electroporated an I-SceI expression plasmid along with HaeIII fragments of X174 genomic DNA. We report that 18 out of 132 tk-deficient clones recovered had captured DNA fragments, and 14 DNA capture events involved one or more fragments of X174 DNA. Microhomology existed at most junctions between X174 DNA and genomic sequences. Our work suggests that virtually any extrachromosomal DNA molecule may be recruited for the patching of DSBs in a mammalian genome.     

Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G²). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.     

Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells

Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1^– cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3′ tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3′ tails after treatment with the rare-cutting endonuclease I-SceI. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3′ tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3′ tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SceI-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SceI-induced double-strand breaks.     

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.     

Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum

Flavobacterium psychrophilum is a Gram-negative fish pathogen that causes important economic losses in aquaculture worldwide. Although the genome of this bacterium has been determined, the function and relative importance of genes in relation to virulence remain to be established. To investigate their respective contribution to the bacterial pathogenesis, effective tools for gene inactivation are required. In the present study, a markerless gene deletion system has been successfully developed for the first time in this bacterium. Using this method, the F. psychrophilum fcpB gene, encoding a predicted cysteine protease homologous to Streptococcus pyogenes streptopain, was deleted. The developed system involved the construction of a conjugative plasmid that harbors the flanking sequences of the fcpB gene and an I-SceI meganuclease restriction site. Once this plasmid was integrated in the genome by homologous recombination, the merodiploid was resolved by the introduction of a plasmid expressing I-SceI under the control of the fpp2 F. psychrophilum inducible promoter. The resulting deleted fcpB mutant presented a decrease in extracellular proteolytic activity compared to the parental strain. However, there were not significant differences between their LD₅₀ in an intramuscularly challenged rainbow trout infection model. The mutagenesis approach developed in this work represents an improvement over the gene inactivation tools existing hitherto for this “fastidious” bacterium. Unlike transposon mutagenesis and gene disruption, gene markerless deletion has less potential for polar effects and allows the mutation of virtually any non-essential gene or gene clusters.     

I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH₂-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 × 10⁻⁶) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.     

An xrcc4 defect or Wortmannin stimulates homologous recombination specifically induced by double-strand breaks in mammalian cells

Non-homologous end joining (NHEJ) and homologous recombination (HR) are two alternative/competitor pathways for the repair of DNA double-strand breaks (DSBs). To gain further insights into the regulation of DSB repair, we detail here the different HR pathways affected by (i) the inactivation of DNA-PK activity, by treatment with Wortmannin, and (ii) a mutation in the xrcc4 gene, involved in a late NHEJ step, using the XR-1 cell line. Here we have analyzed not only the impact of NHEJ inactivation on recombination induced by a single DSB targeted to the recombination substrate (using I-SceI endonuclease) but also on γ-ray- and UV-C-induced and spontaneous recombination and finally on Rad51 foci formation, i.e. on the assembly of the homologous recombination complex, at the molecular level. The results presented here show that in contrast to embryonic stem cells, the xrcc4 mutation strongly stimulates I-SceI-induced HR in adult hamster cells. More precisely, we show here that both single strand annealing and gene conversion are stimulated. In contrast, Wortmannin does not affect I-SceI-induced HR. In addition, γ-ray-induced recombination is stimulated by both xrcc4 mutation and Wortmannin treatment in an epistatic-like manner. In contrast, neither spontaneous nor UV-C-induced recombination was affected by xrcc4 mutation, showing that the channeling from NHEJ to HR is specific to DSBs. Finally, we show here that xrcc4 mutation or Wortmannin treatment results in a stimulation of Rad51 foci assembly, thus that a late NHEJ step is able to affect Rad51 recombination complex assembly. The present data suggest a model according to which NHEJ and HR do not simply compete for DSB repair but can act sequentially: a defect in a late NHEJ step is not a dead end and can make DSB available for subsequent Rad51 recombination complex assembly.     

Deletion of a trypanosome telomere leads to loss of silencing and progressive loss of terminal DNA in the absence of cell cycle arrest

Eukaryotic chromosomes are capped with telomeres which allow complete chromosome replication and prevent the ends from being recognized by the repair machinery. The African trypanosome, Trypanosoma brucei, is a protozoan parasite where antigenic variation requires reversible silencing of a repository of telomere-adjacent variant surface glycoprotein (VSG) genes. We have investigated the role of the telomere adjacent to a repressed VSG. In cells lacking telomerase, the rate of telomere-repeat loss appeared to be inversely proportional to telomere length. We therefore constructed strains in which a single telomere could be immediately removed by conditional I-SceI meganuclease cleavage. Following telomere deletion, cells maintain and segregate the damaged chromosome without repairing it. These cells continue to proliferate at the normal rate but progressively lose terminal DNA at the broken end. Although sirtuin-dependent repression is lost along with the telomere, VSG-silencing is preserved. The results provide direct evidence for telomere-dependent repression but suggest a telomere-independent mode of VSG-silencing. They also indicate the absence of a telomere-loss checkpoint in T. brucei.     

The <Emphasis Type="Italic">GAL10</Emphasis> Gene is Located 40 kbp Away from the <Emphasis Type="Italic">GAL7</Emphasis>-<Emphasis Type="Italic">GAL1</Emphasis> Region in the Yeast <Emphasis Type="Italic">Kazachstania naganishii</Emphasis>

Of the genes involved in galactose metabolism, GAL7, GAL10, and GAL1 are tightly linked in this order on chromosome II in Saccharomyces cerevisiae. While several species of the order Saccharomycetales have similar gene organization, Kazachstania naganishii is unique, in which GAL7 and GAL1 are close to each other whereas GAL10 is substantially apart from them on chromosome XI. In this study, we inserted the recognition sequence of I-SceI homing-endonuclease into GAL10 and also into the intervening segment of GAL7-GAL1. By cleaving chromosome DNA of the gene-manipulated strain with I-SceI, we obtained evidence that chromosome XI (610 kbp) was replaced with three fragments (305, 265, and 40 kbp). Using appropriate probes, we further found that GAL10 was about 40 kbp apart from the GAL7-GAL1 cluster and that orientation of GAL10 was reversed comparing to the S. cerevisiae counter part. We, therefore, contend that comparison of the organization of the GAL cluster among Saccharomycetales is of importance to elucidate evolution of chromosomes and that the experimental scheme developed in this study is useful for this line of investigation.     

Genome mapping ofClostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne

The intron-encoded endonuclease I-CeuI fromChlamydomonas eugametos was shown to cleave the circular chromosomes of allClostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins,β, ε, and?, in addition to the enterotoxin andλ-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins,? andμ, were missing.     

Processing by MRE11 is involved in the sensitivity of subtelomeric regions to DNA double-strand breaks

The caps on the ends of chromosomes, called telomeres, keep the ends of chromosomes from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion. However, subtelomeric regions are sensitive to DSBs, which in normal cells is responsible for ionizing radiation-induced cell senescence and protection against oncogene-induced replication stress, but promotes chromosome instability in cancer cells that lack cell cycle checkpoints. We have previously reported that I-SceI endonuclease-induced DSBs near telomeres in a human cancer cell line are much more likely to generate large deletions and gross chromosome rearrangements (GCRs) than interstitial DSBs, but found no difference in the frequency of I-SceI-induced small deletions at interstitial and subtelomeric DSBs. We now show that inhibition of MRE11 3′–5′ exonuclease activity with Mirin reduces the frequency of large deletions and GCRs at both interstitial and subtelomeric DSBs, but has little effect on the frequency of small deletions. We conclude that large deletions and GCRs are due to excessive processing of DSBs, while most small deletions occur during classical nonhomologous end joining (C-NHEJ). The sensitivity of subtelomeric regions to DSBs is therefore because they are prone to undergo excessive processing, and not because of a deficiency in C-NHEJ in subtelomeric regions.     

An Enhanced Gene Targeting Toolkit for Drosophila: Golic+

Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer “genes” in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report gene targeting during oogenesis with lethality inhibitor and CRISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressor-based lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.