Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment

Caenorhabditis elegans is an important model organism for modern biologic research. An essential aspect of C. elegans research is the production of transgenic animals for study. These are often generated via microinjection, but biolistic bombardment has become increasingly popular. However, many of the plasmids previously generated for use in microinjection are not readily used for bombardment due to the lack of a convenient marker. The unc-119 gene is often used as a marker since unc-119 rescue can be observed at low magnification, allowing rescued animals to be easily distinguished from the larger number of non-rescued animals. Here we report the use of homologous recombination in Escherichia coli as a method to insert a cassette containing the unc-119 gene into commonly used plasmids at the site of the ampicillin resistance gene which is simpler than other methods like subcloning. These cassettes are flanked by regions homologous to the 5′ and 3′ ends of the ampicillin resistance gene and contain either the unc-119 gene and the kanamycin resistance gene or a unc-119:mCherry fusion gene and the kanamycin resistance gene. The resulting plasmids may be used for biolistic bombardment to yield animals that display unc-119 rescue, and also express the recipient plasmid transgene.     

Homologous gene targeting in Caenorhabditis elegans by biolistic transformation

Targeted homologous recombination is a powerful approach for genome manipulation that is widely used for gene alteration and knockouts in mouse and yeast. In Caenorhabditis elegans, several methods of target-selected mutagenesis have been implemented but none of them provides the opportunity of introducing exact predefined changes into the genome. Although anecdotal cases of homologous gene targeting in C.elegans have been reported, no practical technique of gene targeting has been developed so far. In this work we demonstrate that transformation of C.elegans by microparticle bombardment (biolistic transformation) can result in homologous recombination between introduced DNA and the chromosomal locus. We describe a scaled up version of biolistic transformation that can be used as a method for homologous gene targeting in the worm.     

A Co-CRISPR Strategy for Efficient Genome Editing in Caenorhabditis elegans

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.     

The nphp-2 and arl-13 Genetic Modules Interact to Regulate Ciliogenesis and Ciliary Microtubule Patterning in C. elegans

Cilia are microtubule-based cellular organelles that mediate signal transduction. Cilia are organized into several structurally and functionally distinct compartments: the basal body, the transition zone (TZ), and the cilia shaft. In vertebrates, the cystoprotein Inversin localizes to a portion of the cilia shaft adjacent to the TZ, a region termed the “Inversin compartment” (InvC). The mechanisms that establish and maintain the InvC are unknown. In the roundworm C. elegans, the cilia shafts of amphid channel and phasmid sensory cilia are subdivided into two regions defined by different microtubule ultrastructure: a proximal doublet-based region adjacent to the TZ, and a distal singlet-based region. It has been suggested that C. elegans cilia also possess an InvC, similarly to mammalian primary cilia. Here we explored the biogenesis, structure, and composition of the C. elegans ciliary doublet region and InvC. We show that the InvC is conserved and distinct from the doublet region. nphp-2 (the C. elegans Inversin homolog) and the doublet region genes arl-13, klp-11, and unc-119 are redundantly required for ciliogenesis. InvC and doublet region genes can be sorted into two modules—nphp-2+klp-11 and arl-13+unc-119—which are both antagonized by the hdac-6 deacetylase. The genes of this network modulate the sizes of the NPHP-2 InvC and ARL-13 doublet region. Glutamylation, a tubulin post-translational modification, is not required for ciliary targeting of InvC and doublet region components; rather, glutamylation is modulated by nphp-2, arl-13, and unc-119. The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes. NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC. We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.     

Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.     

Improved techniques for endogenous epitope tagging and gene deletion in Toxoplasma gondii

Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii.     

Genetic Organization in CAENORHABDITIS ELEGANS: Fine-Structure Analysis of the unc-22 Gene

Fine-structure analysis of the unc-22 gene of Caenorhabditis elegans has revealed a number of sites that are separable by recombination. Eight new ethyl methanesulfonate-induced recessive mutations of the unc-22 gene have been isolated. Using these new alleles, as well as e66, a number of separable sites have been identified and positioned relative to one another. The map distances obtained are found to be comparable to those associated with intragenic recombination in Drosophila melanogaster, indicating that genetic fine-structure analysis is feasible in Caenorhabditis elegans. Evidence of possible gene conversion is presented. A preliminary estimate of the unc-22 gene size is 2.4 x 10^-2 map units.     

Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs

Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.     

Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli

The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc^R) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc^R. A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems.     

Progress in lactic acid bacterial phage research

Background

The cyanobacterium Synechocystis sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids.

Results

In this study, we present a marker-less gene deletion and replacement strategy in Synechocystis sp. PCC 6803 which needs only a single transformation step. The method utilizes an nptI-sacB double selection cassette and exploits the ability of the cyanobacterium to undergo two successive genomic recombination events via double and single crossing-over upon application of appropriate selective procedures.

Conclusions

By reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.     

Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.     

The DH-PH region of the giant protein UNC-89 activates RHO-1 GTPase in Caenorhabditis elegans body wall muscle

Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing a DH domain followed by a PH domain at its N terminus, which is characteristic of guanine nucleotide exchange factor proteins for Rho GTPases. To obtain evidence that the DH-PH region has activity toward specific Rho family small GTPases, we conducted an experiment using the yeast three-hybrid system. The DH-PH region of UNC-89 has exchange activity for RHO-1 (C. elegans RhoA), but not for CED-10 (C. elegans Rac), MIG-2 (C. elegans RhoG), or CDC-42 (C. elegans Cdc42). The DH domain alone has similar activity for RHO-1. An in vitro binding assay demonstrates interaction between the DH-PH region of UNC-89 and each of the C. elegans Rho GTPases. Partial knockdown of rho-1 in C. elegans adults showed a pattern of disorganization of myosin thick filaments similar to the phenotype caused by unc-89 (su75), a mutant allele in which all of the isoforms containing the DH-PH region are missing. Taken together, we propose a model in which the DH-PH region of UNC-89 activates RHO-1 GTPase for organization of myosin filaments in C. elegans muscle cells.     

Transposon-mediated generation of targeting vectors for the production of gene knockouts

Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of gene targeting technology include gene replacement (knockins) and conditional vectors which allow for the generation of inducible or tissue-specific gene-targeting events. The assembly of gene-targeting vectors is generally a laborious process requiring considerable technical skill. The procedures presented here report the application of transposons as tools for the construction of targeting vectors. Two mini-Mu transposons were sequentially inserted by in vitro transposition at each side of the region targeted for deletion. One such transposon carries an antibiotic resistance marker suitable for selection in mammalian cells. A deletion is then generated between the two transposons either by LoxP-induced recombination or by restriction digestion followed by ligation. This deletion removes part of both transposons plus the targeted region in between, leaving a transposon carrying the selectable marker flanked by two arms which are homologous to the targeted gene. Targeting vectors constructed using these transposons were electroporated into embryonic stem cells and shown to be effective in gene-targeting events.     

Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens

In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates predominantly by homologous recombination with its genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology length and targeting frequency for replacement vectors (a selectable marker flanked by homologous DNA). Overall homology of only 1 kb is sufficient to achieve a 50% yield of targeted transformants. Targeting may occur through homologous recombination in one arm, accompanied by non-homologous end-joining by the other arm of the vector, or by allele replacement following two homologous recombination events. Allele replacement frequency depends on the symmetry of the targeting vector, being proportional to the length of the shorter arm. Allele replacement may involve insertion of multiple copies of the transforming DNA, accompanied by ectopic insertions at non-homologous sites. Single-copy and single insertions at targeted loci (targeted gene replacements, ‘TGR’) occur with a frequency of 7–20% of all transformants when the minimum requirements for allele replacement are met. Homologous recombination in Physcomitrella is substantially more efficient than in any multicellular eukaryote, recommending it as the outstanding model for the study of homologous recombination in plants.     

Targeted disruption of the TGA3 locus in Arabidopsis thaliana

A major drawback to study gene functions in plant systems is the lack of an effective gene knockout strategy. With a large number of plant genes isolated and the accelerating pace by which this collection is growing, the need for their functional analyses at the whole plant level has become increasingly urgent. Here evidence is reported for the first successful disruption of a non-selectable gene in Arabidopsis thaliana by creating a mutant of the TGA3 locus via targeted insertion of the bacterial neo gene conferring kanamycin (Km) resistance. A β-glucuronidase (GUS) expression unit outside the region of homology was used as a screenable marker to distinguish homologous recombination events from those of ectopic insertions. PCR amplification coupled with Southern blot screening identified two putative homologous recombination events among 2580 Km^r calli. One callus line was subsequently isolated and the structure of the targeted TGA3 allele confirmed by Southern blot analyses. This study demonstrates the feasibility of targeting a non-selectable locus in Arabidopsis. Combined with future improvements in negative selection strategies and efficient, transformation methodologies, gene replacement studies in plants could become a routine technique.     

An efficient method for gene disruption in Neurospora crassa

The frequency with which transforming DNA undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. In such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. A protocol is presented for efficient isolation of Neurospora crassa strains carrying a definitive null allele in a target gene. The protocol relies on the presence of a selectable marker flanking a disrupted plasmid-borne copy of the gene, and in the case presented led to a seven-fold enrichment for putative homologous site replacement events. In addition, a polymerase chain reaction assay is utilized for rapid identification of homologous recombinants among the remaining candidates. This protocol was used to identify 3 isolates, out of 129 primary transformants, which have a disruption in the Neurospora ccg-1 gene. The method should be applicable to a variety of fungal systems in which two selectable markers can be expressed, including those in which homologous recombination rates are too low to allow easy identification of homologous site insertions by the more traditional molecular method of Southern analysis. In addition to disrupting target genes for the purpose of generating null mutations, this method is useful for the targeting of reporter gene fusions to a native chromosomal site for the purpose of studying gene regulation.     

A Conditional Knockout Toolkit for Caenorhabditis elegans Based on the Cre/loxP Recombination

Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.     

Molecular Characterization of a Soybean Cyst Nematode (Heterodera glycines) Homolog of unc-87

In Caenorhabditis elegans the unc-87 gene encodes a protein that binds to actin at the I band and is important in nematodes for maintenance of the body-wall muscle. Caenorhabditis elegans mutant phenotypes of unc-87 exhibit severe paralysis in larvae and limp paralysis in the adult. We cloned and characterized a full-length cDNA representing a Heterodera glycines homolog of the unc-87 gene from C. elegans that encodes a protein that contains a region of seven repeats similar to CLIK-23 from C-elegans and has 81% amino acid identity with that of C. elegans unc-87 variant A. In the EST database clones labeled "unc-87'''' encode mainly the 3'' portion of unc-87, while clones labeled "calponin homolog OV9M'''' contain mainly DNA sequence representing the 5'' and middle transcribed regions of unc-87. A 1770 nucleotide cDNA encoding H. glycines unc-87 was cloned and encodes a predicted UNC-87 protein product of 375 amino acids. The expression of unc-87 was determined using RT-PCR and, in comparison to its expression in eggs, unc-87 was expressed 6-fold higher in J2 juveniles and 20-fold and 13-fold (P = 0.05) higher in nematodes 15 and 30 days after inoculation, respectively. In situ hybridization patterns confirmed the expression patterns observed with RT-PCR.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^?) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^? transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^?. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.