Double-crossover Event Research Articles

Novel Double Fusion (βδβ) Hemoglobin Variant with Comparison to Lepore Hemoglobins

Lepore hemoglobins (Hbs) such as Hb Lepore-Boston-Washington [δ(1-87) β(116-146)], Hb Lepore-Baltimore [δ(1-50) β(86-146)], Hb Lepore-Hollandia [δ(1-22) β(50-146)] are δβ gene fusion/deletion variants (ε- Gγ- Aγ-[δβ] v )(Figure 1). They are controlled by the weak δ-globin gene promoter and synthesized at approximately 10-15% of hemoglobin fractions. This results in a similar phenotype as β +-thalassemia (microcytosis and hypochromia ± mild anemia). Anti-Lepore hemoglobins such as Hb P-Nilotic (β(1-22) δ(50-146) are βδ gene fusion/duplication variants (ε- Gγ- Aγ-δ-[βδ] v-β) associated with normal CBC values. Herein we describe a novel βδβ double fusion variant (ε- Gγ- Aγ-δ-[βδβ] v). Due to high homology between the δ and β genes, this double fusion event encodes an identical protein to Hb Lepore-Hollandia, albeit linked to the β-promoter and therefore expressed at ~40% of hemoglobin fractions, which would be expected to enhance any intrinsic properties of the variant. This novel hemoglobin variant is named Hb Lepore Rochester-MN. Two cases have been identified (Table 1). Case 1 was a 10-month-old Caucasian/Native American infant referred for an abnormal newborn screen and a reported family history of Hb G-Coushatta (β22, GAA>GCA, Glu>Ala; HGVS: c.68A>C, p.E23A). Case 2 was a 3-month-old infant referred for an abnormal newborn screen. CBCs were normal for age including MCV. Cation-exchange high performance liquid chromatography (HPLC) showed a shouldered peak in the Hb A 2 window typical for Hb Lepore (elution time: 3.41-3.46 mins), although the variant percentage was higher than expected, mimicking Hb G-Coushatta (expected elution time: 3.33 mins). Capillary electrophoresis identified a peak in the D zone (Z(D) zone), which is expected for Hb Lepore or Hb G-Coushatta. Intact mass spectrometry was consistent with Hb Lepore-Hollandia at 15836 amu, differing from Hb G-Coushatta (Glu>Ala, 15809 amu). Multiplex ligand probe amplification (MLPA) was normal because the highly variable fusion regions are unsuitable for probe placement. Sanger sequencing of the β-globin gene identified multiple single base substitutions which were challenging to interpret but matched the sequence expected for a complex double crossover event (NM_000518.4:c.28_68delins41 (p.Ser10_Glu23delins14TAVNALWGKVNVDA). The first crossover is located between c.-29 and c.9, the second crossover is located between c.69 and c.92+17. Long-read sequencing was performed (Sequel, Pacific Biosciences) which detected the multiple missense mutations in cis conformation, as was corroborated by the mass spectrometry data. In summary, Hb Lepore Rochester-MN represents a novel double fusion [βδβ] variant class that will pose a diagnostic challenge for laboratories. Although it is likely a result of sequential crossover events (deletion and duplication) between Lepore and anti-Lepore variants, MLPA analysis is normal due to technical constraints and high homology. DNA sequencing (both Sanger and long read methods) yielded complex results that require sophisticated analysis and correlation with protein data including intact mass spectrometry. HPLC and CE data including variant percentage are very similar to Hb G-Coushatta. In addition, although resulting in an identical protein, the variant should not be mistaken for Hb Lepore-Hollandia, which is produced at lower levels and causes a thalassemic phenotype. Hb Lepore Rochester-MN is controlled by the β-promoter and therefore is associated with approximately 40% variant and apparently normal CBC values in the two infants.

Creation of a new Drosophila melanogaster X chromosome balancer, First Multiple 8 ( FM8 ), using the CRISPR/Cas9 gene-editing system.

Balancer chromosomes contain multiple inversions that work to suppress crossing over and prevent recovery of most recombinant chromosomes, allowing for alleles to be kept long-term without selection. These balancers are incredible tools, but some alleles within larger inverted segments are still lost to rare double crossover events between the balanced and balancer chromosomes. This study details a new methodology of producing balancer chromosomes using CRISPR/Cas9 gene editing technology to create new inversions within the largest segment of the common X chromosome balancer, FM7c. We were able to create a new X chromosome balancer, FM8, and anticipate that this process can be used to not only create other balancers in Drosophila melanogaster, but other model organisms as well.

Double Crossover Approach to Inactivate Target Gene in Cyanobacteria.

Anabaena sp. PCC7120 (hereafter Anabaena 7120) is a nitrogen-fixing, filamentous cyanobacterium. Given its diverse metabolism, it serves as an excellent model organism, particularly for studying cell differentiation, nitrogen fixation, photosynthesis, production of high-value chemicals, and synthetic biology. Gene knockout is a common approach to assess the function of gene products through assessing phenotypic loss of function. In the method described here, a double crossover approach is used to inactivate a target gene or target genes in Anabaena 7120. This method involves replicating the gene(s) from the wild-type genomic DNA and inserting them into an integrative plasmid vector. An internal portion of the genes may be removed and replaced with a GFP-Spectinomycin (gfp-sp) cassette. The plasmid is then introduced into Anabaena 7120 where a double crossover event occurs between the wild-type chromosome and the cargo plasmid, effectively replacing the wild-type gene with the disrupted gene from the plasmid. The gfp-sp cassette combined with the sacB gene serve as positive selection to identify double crossover mutants (Caiand Wolk(1990), 172(6):3138-3145, J.Bacteriol). Finally, the functional genes are cloned into another replicating plasmid vector to produce a cargo plasmid, which is conjugatively introduced into the mutant for a complementation test. By comparing the phenotypes among the wild-type, mutant, and complement, one should see a loss of function in the mutant which is recovered in the complement, thereby defining the function of the target gene. The double crossover approach described here for Anabaena PCC 7120 may be broadly applicable to the study of gene function in cyanobacteria and other prokaryotic organisms.

The Role of Chromatid Interference in Determining Meiotic Crossover Patterns

Plants, like all sexually reproducing organisms, create genetic variability by reshuffling parental alleles during meiosis. Patterns of genetic variation in the resulting gametes are determined by the independent assortment of chromosomes in meiosis I and by the number and positioning of crossover (CO) events during meiotic recombination. On the chromosome level, spatial distribution of CO events is biased by multiple regulatory mechanisms, such as CO assurance, interference and homeostasis. However, little is known about how multiple COs are distributed among the four chromatids of a bivalent. Chromatid interference (CI) has been proposed as a regulatory mechanism that biases distribution of multiple COs toward specific chromatid partners, however, its existence has not been well-studied and its putative mechanistic basis remains undescribed. Here, we introduce a novel method to quantitatively express CI, and take advantage of available tetrad-based genotyping data from Arabidopsis and maize male meiosis to quantify CI effects on a genome-wide and chromosomal scale. Overall, our analyses reveal random involvement of sister chromatids in double CO events across paired chromosomes, indicating an absence of CI. However, on a genome-wide level, CI was found to vary with physical distance between COs, albeit with different effects in Arabidopsis and maize. While effects of CI are minor in Arabidopsis and maize, the novel methodology introduced here enables quantitative interpretation of CI both on a local and genome-wide scale, and thus provides a key tool to study CI with relevance for both plant genetics and crop breeding.

Molecular Biology Methods in Streptomyces rimosus, a Producer of Oxytetracycline.

Streptomyces rimosus is used for production of the broad-spectrum antibiotic oxytetracycline (OTC). S. rimosus belongs to Actinomyces species, a large group of microorganisms that produce diverse set of natural metabolites of high importance in many aspects of our life. In this chapter, we describe specific molecular biology methods and a classical homologous recombination approach for targeted in-frame deletion of a target gene or entire operon in S. rimosus genome. The presented protocols will guide you through the design of experiment and construction of homology arms and their cloning into appropriate vectors, which are suitable for gene-engineering work with S. rimosus. Furthermore, two different protocols for S. rimosus transformation are described including detailed procedure for targeted gene replacement via double crossover recombination event. Gene deletion is confirmed by colony PCR, and colonies are further characterized by cultivation and metabolite analysis. As the final step, we present in trans complementation of the deleted gene, to confirm functionality of the engineering approach achieved by gene disruption. A number of methodological steps and protocols are optimized for S. rimosus strains including the use of the selected reporter genes. Protocols described in this chapter can be applied for studying function of any individual gene product in diverse OTC-producing Streptomyces rimosus strains.

A New Suite of Allelic-Exchange Vectors for the Scarless Modification of Proteobacterial Genomes.

Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through homologous recombination remains an important tool for genetic analysis. Currently, sacB-based vector systems are often used for allelic exchange, but counterselection escape, which prevents isolation of cells with the desired mutation, occasionally limits their utility. To circumvent this, we engineered a series of "pTOX" allelic-exchange vectors. Each plasmid encodes one of a set of inducible toxins, chosen for their potential utility in a wide range of medically important proteobacteria. A codon-optimized rhaS transcriptional activator with a strong synthetic ribosome-binding site enables tight toxin induction even in organisms lacking an endogenous rhamnose regulon. Expression of the gene encoding blue AmilCP or magenta TsPurple nonfluorescent chromoprotein facilitates monitoring of successful single- and double-crossover events using these vectors. The versatility of these vectors was demonstrated by deleting genes in Serratia marcescens, Escherichia coli O157:H7, Enterobacter cloacae, and Shigella flexneri Finally, pTOX was used to characterize the impact of disruption of all combinations of the 3 paralogous S. marcescens peptidoglycan amidohydrolases on chromosomal ampC β-lactamase activity and the corresponding β-lactam antibiotic resistance. Mutation of multiple amidohydrolases was necessary for high-level ampC derepression and β-lactam resistance. These data suggest why β-lactam resistance may emerge during treatment less frequently in S. marcescens than in other AmpC-producing pathogens, like E. cloacae Collectively, our findings suggest that the pTOX vectors should be broadly useful for genetic engineering of Gram-negative bacteria.IMPORTANCE Targeted modification of bacterial genomes is critical for genetic analysis of microorganisms. Allelic exchange is a technique that relies on homologous recombination to replace native loci with engineered sequences. However, current allelic-exchange vectors often enable only weak selection for successful homologous recombination. We developed a suite of new allelic-exchange vectors, pTOX, which were validated in several medically important proteobacteria. They encode visible nonfluorescent chromoproteins that enable easy identification of colonies bearing integrated vectors and permit stringent selection for the second step of homologous recombination. We demonstrate the utility of these vectors by using them to investigate the effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on β-lactam antibiotic resistance.

28. KARYOMAPPING USING WHOLE GENOME SEQUENCING DATA: PRECLINICAL VALIDATION OF A PERSONALISED APPROACH TO PGT-M

Introduction Karyomapping utilises microarray technology to track informative single nucleotide polymorphisms (SNPs) and assign haplotypes to embryos without the need for direct mutation detection (Handyside et al. 2010). Although suitable for most couples undergoing PGT-M, the use of a fixed set of SNPs may provide inadequate information in certain genetic regions or in consanguineous couples (Konstantinidis et al. 2015). Whole genome sequencing (WGS) has the potential to individualise informative SNP detection and perform concurrent direct mutation detection. However, no studies have evaluated the potential for application of the karyomapping algorithm to WGS data. Material and Methods The Platinum Genomes dataset ( https://sapac.illumina.com/platinumgenomes.html ) was analysed as a model for the application of karyomapping to WGS data. Variant call format (VCF) files were analysed according to the principles of karyomapping to create a map of informative biallelic SNPs in each grandparental haplotype. A model of embryo biopsy was created by diluting DNA from one of the family members (NA12878) to 30pg and whole genome amplification was performed with the Repli-G single cell kit (Qiagen, Hilden, Germany) as a model of trophectoderm biopsy. Whole genome sequencing was performed in triplicate on a high output NextSeq 2500 run (Illumina, California, USA) at average 15x coverage and the number of informative SNPs detected compared to the karyomapping SNP microarray. Comparison of variant calling methods was performed using the Genome Analysis Toolkit v3.4 (Broad Institute, Massachusetts, USA) and DeepVariant (Google, California, USA). Results Analysis of WGS data from the Platinum Genomes family revealed a total of 2,338,158 informative SNPs distributed evenly between grandparental haplotypes, of which 85,053 (3.6%) would have been detectable using the karyomapping SNP array. Analysis of the karyomapping window around common genes requiring PGT-M demonstrated an average 32x increase in the number of targets available for analysis in a PGT-M cycle. Analysis of 15x coverage WGS data in a model of embryo biopsy demonstrated an average 279,851 informative SNPs detected using standard variant calling and an average 285,107 using AI-based variant calling. The median distance between informative SNPs was reduced from 26,782 nucleotides to 1,212 nucleotides when comparing WGS-based karyomapping to SNP array-based karyomapping. Conclusions This study provides preclinical validation for the application of the karyomapping algorithm to WGS data. The markedly increased number of informative SNPs will allow successful feasibility studies for all couples presenting for PGT-M. The decreased distance between informative SNPs detected in a model embryo biopsy will improve diagnostic accuracy, particularly in detecting small double crossover events and reducing the number of cycles in which inadequate informative markers have been detected in the embryo sample. Finally, the use of WGS for diagnostic purposes, especially for rare and undiagnosed genetic disease, will allow the creation of a high resolution digital karyomap for couples with an affected child and improve the transition to IVF and PGT-M.

A Single-Plasmid Genome Editing System for Metabolic Engineering of Lactobacillus casei.

Genome engineering of Lactobacillus casei, an important industrial microorganism for dairy fermented product, currently relies on inefficient and time-consuming double crossover events. In this study, we developed an easy-to-use genome engineering strategy for metabolic engineering of L. casei for acetoin production. Plasmid pMSP456-Cre, that contains prophage recombinase operon LCABL_13040-50-60 driven by the nisin-controlled inducible expression (NICE) system and the site-specific recombinase gene cre under the control of the promoter of the lactose operon from L. casei, was constructed. Using this plasmid, integration of a hicD3 gene linear donor cassette (up-lox66-cat-lox71-down) was catalyzed by the LCABL_13040-50-60 recombinase and the cat gene was excised by the Cre/lox system with an efficiency of 60%. To demonstrate this system for sequential and iterative knocking out genes in L. casei, another three genes (pflB, ldh and pdhC) related to acetoin production were deleted with the efficiencies of 60, 40, and 60%, respectively. The yielding quadruple mutant could produce a ∼18-fold higher amount of acetoin than the wild-type and converted 59.8% of glucose to acetoin in aerobic. Therefore, these results proved this simple genome engineering strategy have potential in metabolic engineering of L. casei for production of high value-added metabolites.

Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9

BackgroundTargeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired.ResultsWe generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17–0.45%) that was used to knock in an approximately 5-kb long DNA fragment.ConclusionWe developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application.

An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA.

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30mg/l.

The Molecular and Genetic Characterization of Second Chromosome Balancers in Drosophila melanogaster.

Balancer chromosomes are multiply inverted and rearranged chromosomes used in Drosophila melanogaster for many tasks, such as maintaining mutant alleles in stock and complex stock construction. Balancers were created before molecular characterization of their breakpoints was possible, so the precise locations of many of these breakpoints are unknown. Here, we report or confirm the positions of the 14 euchromatic breakpoints on the 2nd chromosome balancers SM1, SM5, CyO, and SM6a. This total includes three breakpoints involved in a complex rearrangement on SM5 that is associated with the duplication of two genomic regions. Unbiased sequencing of several balancers allowed us to identify stocks with incorrectly identified balancers as well as single and double crossover events that had occurred between 2nd chromosome balancers and their homologs. The confirmed crossover events that we recovered were at least 2 Mb from the closest inversion breakpoint, consistent with observations from other balancer chromosomes. Balancer chromosomes differ from one another both by large tracts of sequence diversity generated by recombination and by small differences, such as single nucleotide polymorphisms (SNPs). Therefore, we also report loss-of-function mutations carried by these chromosomes and unique SNP and InDel polymorphisms present on only single balancers. These findings provide valuable information about the structure of commonly used 2nd chromosome balancers and extend recent work examining the structure of X and 3rd chromosome balancers. Finally, these observations provide new insights into how the sequences of individual balancers have diverged over time.

High-throughput system for quantifying and characterizing homologous recombination in Chlamydomonas reinhardtii

In the green alga Chlamydomonas reinhardtii, introduced DNA fragments predominantly insert randomly into the nuclear genome by non-homologous recombination (NHR), often resulting in highly variable phenotypes between transformants. Homologous recombination (HR) can occur in C. reinhardtii but at very low frequency, and is often accompanied by insertions, deletions, and/or rearrangements at the recombination site. To benchmark the frequency and characterize the nature of HR integrations in C. reinhardtii, we developed a system for detecting and characterizing HR events that includes three intact markers and one split marker as well a novel design element: utilizing a long intron as the homology region. In this study we demonstrate that this system meets the following criteria: accommodates high-throughput screening; provides a high-fidelity phenotype for detecting HR without false positives from reversion or locus heterogeneity; allows and captures both single- and double-crossover HR events; reports HR and NHR rates from a single transformation; and allows characterization of imprecise recombination or rearrangement at the integration site. Using this system we reproducibly determined the HR rate in our recipient strain of C. reinhardtii and characterized a number of recombinants by restriction digests and sequencing of PCR amplified recombination junctions to show that both double and single crossover events were recovered and that integration occurred both via perfect and imperfect (i.e. accompanied by insertions, deletions, and rearrangements) HR. This system is valuable for systematically testing approaches for increasing HR efficiency and accuracy.

Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains.

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget's disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum

Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. Here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9 to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecT for efficient curing such that multiple gene targets can be done iteratively and final strains will be free of plasmids. To demonstrate the system, seven different mutants were constructed within two weeks to study the combinatorial deletion effects of three different genes on the production of γ-aminobutyric acid, an industrially relevant chemical of much interest. This genome engineering strategy will expedite metabolic engineering of C. glutamicum.

Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.

BackgroundGeobacillus thermoglucosidasius is a thermophilic, natural ethanol producer and a potential candidate for commercial bioethanol production. Previously, G. thermoglucosidasius has been genetically modified to create an industrially-relevant ethanol production strain. However, creating chromosomal integrations and deletions in Geobacillus spp. is laborious. Here we describe a new technique to create marker-less mutations in Geobacillus utilising a novel homologous recombination process.ResultsOur technique incorporates counter-selection using β-glucosidase and the synthetic substrate X-Glu, in combination with a two-step homologous recombination process where the first step is a selectable double-crossover event that deletes the target gene. We demonstrate how we have utilised this technique to delete two components of the proteinaceous shell of the Geobacillus propanediol-utilization microcompartment, and simultaneously introduce an oxygen-sensitive promoter in front of the remaining shell-component genes and confirm its functional incorporation.ConclusionThe selectable deletion of the target gene in the first step of our process prevents re-creation of wild-type which can occur in most homologous recombination techniques, circumventing the need for PCR screening to identify mutants. Our new technique therefore offers a faster, more efficient method of creating mutants in Geobacillus.

Harnessing The Power Of Recombineering To Interrogate The Virulome Of Escherichia albertii

The causal link between a pathogen’s gene and virulence can be established by fulfilling Molecular Koch’s Postulates, which, in turn, requires the engineering of targeted mutations in the predicted gene of interest. Within the past decade, the paradigm-shifting and revolutionary technology of recombineering has enabled bacteriologists to construct virtually every conceivable mutation in many enteric bacteria. Recombineering exploits the function of bacteriophage-encoded recombinases to bring about allelic replacement. In recombineering, a linear double stranded or single stranded DNA molecule with terminal homology arms, identical to the region upstream and downstream of a genetic locus of interest, is introduced into hyperrecombinogenic bacteria. Recombinases catalyze the replacement of the endogenous allele with the introduced allele by means of a double crossover event. Using this technique, researchers have methodically dissected the virulence repertoire of the attaching and effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohermorrhagic E. coli (EHEC), and Citrobacter rodentium . In contrast, the virulome of the most recent family member, E. albertii , remains unmapped, despite metagenomic sequencing revealing that the bacterium possesses an impressive arsenal of virulence determinants. Moreover, numerous retrospective studies have incriminated E. albertii as the etiologic agent of multiple disease outbreaks in both developed and developing countries. Yet, its pathogenic potential remains cryptic. Therefore, it is imperative to initiate studies to interrogate its virulence mechanisms for developing effective interventions. With this in mind, we developed a lambda red-mediated recombineering protocol to identify and characterize virulence genes in the bacterium. The versatility of recombineering for targeted mutagenesis was demonstrated by allelic replacement of multiple unlinked genetic loci, with a noted role in the virulence of related A/E pathogens. Our protocol will enable researchers to mutagenize any gene in E. albertii to have a concrete understanding of its contribution to bacterial physiology and virulence.

Rare recombination events generate sequence diversity among balancer chromosomes in Drosophila melanogaster

Multiply inverted balancer chromosomes that suppress exchange with their homologs are an essential part of the Drosophila melanogaster genetic toolkit. Despite their widespread use, the organization of balancer chromosomes has not been characterized at the molecular level, and the degree of sequence variation among copies of balancer chromosomes is unknown. To map inversion breakpoints and study potential diversity in descendants of a structurally identical balancer chromosome, we sequenced a panel of laboratory stocks containing the most widely used X chromosome balancer, First Multiple 7 (FM7). We mapped the locations of FM7 breakpoints to precise euchromatic coordinates and identified the flanking sequence of breakpoints in heterochromatic regions. Analysis of SNP variation revealed megabase-scale blocks of sequence divergence among currently used FM7 stocks. We present evidence that this divergence arose through rare double-crossover events that replaced a female-sterile allele of the singed gene (sn(X2)) on FM7c with a sequence from balanced chromosomes. We propose that although double-crossover events are rare in individual crosses, many FM7c chromosomes in the Bloomington Drosophila Stock Center have lost sn(X2) by this mechanism on a historical timescale. Finally, we characterize the original allele of the Bar gene (B(1)) that is carried on FM7, and validate the hypothesis that the origin and subsequent reversion of the B(1) duplication are mediated by unequal exchange. Our results reject a simple nonrecombining, clonal mode for the laboratory evolution of balancer chromosomes and have implications for how balancer chromosomes should be used in the design and interpretation of genetic experiments in Drosophila.

Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi).

Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection.

Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus

In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (ΔpyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophilus was inserted into the chromosome of UBOCC-3256 (ΔpyrF) or UBOCC-3260 (ΔpyrF ΔhisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.

Gamete-Type Dependent Crossover Interference Levels in a Defined Region ofCaenorhabditis elegansChromosome V

In certain organisms, numbers of crossover events for any single chromosome are limited (“crossover interference”) so that double crossover events are obtained at much lower frequencies than would be expected from the simple product of independent single-crossover events. We present a number of observations during which we examined interference over a large region of Caenorhabditis elegans chromosome V. Examining this region for multiple crossover events in heteroallelic configurations with limited dimorphism, we observed high levels of crossover interference in oocytes with only partial interference in spermatocytes.