Optimising Homing Endonuclease Gene Drive Performance in a Semi-Refractory Species: The Drosophila melanogaster Experience

Homing endonuclease gene (HEG) drive is a promising insect population control technique that employs meganucleases to impair the fitness of pest populations. Our previous studies showed that HEG drive was more difficult to achieve in Drosophila melanogaster than Anopheles gambiae and we therefore investigated ways of improving homing performance in Drosophila. We show that homing in Drosophila responds to increased expression of HEGs specifically during the spermatogonia stage and this could be achieved through improved construct design. We found that 3′-UTR choice was important to maximise expression levels, with HEG activity increasing as we employed Hsp70, SV40, vasa and βTub56D derived UTRs. We also searched for spermatogonium-specific promoters and found that the Rcd-1r promoter was able to drive specific expression at this stage. Since Rcd-1 is a regulator of differentiation in other species, it suggests that Rcd-1r may serve a similar role during spermatogonial differentiation in Drosophila. Contrary to expectations, a fragment containing the entire region between the TBPH gene and the bgcn translational start drove strong HEG expression only during late spermatogenesis rather than in the germline stem cells and spermatogonia as expected. We also observed that the fraction of targets undergoing homing was temperature-sensitive, falling nearly four-fold when the temperature was lowered to 18°C. Taken together, this study demonstrates how a few simple measures can lead to substantial improvements in the HEG-based gene drive strategy and reinforce the idea that the HEG approach may be widely applicable to a variety of insect control programs.     

The population genetics of using homing endonuclease genes in vector and pest management

Homing endonuclease genes (HEGs) encode proteins that in the heterozygous state cause double-strand breaks in the homologous chromosome at the precise position opposite the HEG. If the double-strand break is repaired using the homologous chromosome, the HEG becomes homozygous, and this represents a powerful genetic drive mechanism that might be used as a tool in managing vector or pest populations. HEGs may be used to decrease population fitness to drive down population densities (possibly causing local extinction) or, in disease vectors, to knock out a gene required for pathogen transmission. The relative advantages of HEGs that target viability or fecundity, that are active in one sex or both, and whose target is expressed before or after homing are explored. The conditions under which escape mutants arise are also analyzed. A different strategy is to place HEGs on the Y chromosome that cause one or more breaks on the X chromosome and so disrupt sex ratio. This strategy can cause severe sex-ratio biases with efficiencies that depend on the details of sperm competition and zygote mortality. This strategy is probably less susceptible to escape mutants, especially when multiple X shredders are used.     

P-element homing is facilitated by engrailed polycomb-group response elements in Drosophila melanogaster

P-element vectors are commonly used to make transgenic Drosophila and generally insert in the genome in a nonselective manner. However, when specific fragments of regulatory DNA from a few Drosophila genes are incorporated into P-transposons, they cause the vectors to be inserted near the gene from which the DNA fragment was derived. This is called P-element homing. We mapped the minimal DNA fragment that could mediate homing to the engrailed/invected region of the genome. A 1.6 kb fragment of engrailed regulatory DNA that contains two Polycomb-group response elements (PREs) was sufficient for homing. We made flies that contain a 1.5 kb deletion of engrailed DNA (en(Δ1.5)) in situ, including the PREs and the majority of the fragment that mediates homing. Remarkably, homing still occurs onto the en(Δ1. 5) chromosome. In addition to homing to en, P[en] inserts near Polycomb group target genes at an increased frequency compared to P[EPgy2], a vector used to generate 18,214 insertions for the Drosophila gene disruption project. We suggest that homing is mediated by interactions between multiple proteins bound to the homing fragment and proteins bound to multiple areas of the engrailed/invected chromatin domain. Chromatin structure may also play a role in homing.     

The evolution of homing endonuclease genes and group I introns in nuclear rDNA

Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.     

Functional lentiviral vectors for xeroderma pigmentosum gene therapy

Xeroderma pigmentosum (XP) is a genetic disease characterized by an autosomal-transmitted genodermatosis involving impaired DNA repair activity, where XP patients present severe sensitivity to sunlight (UVB radiation) and are highly victimized by skin cancer. Complementing XP genes by gene therapy is one potential strategy for helping XP patients. However, current viral-based protocols still lack long-term and stable expression, due to limited post-mitotic infection and gene silencing (in the case of retroviruses) or transient expression and activation of immune response (in the case of adenoviruses). Here we demonstrate that the use of third-generation lentiviral vectors can overcome some of these limitations, rescuing the aberrant phenotype in different categories of the disease (XPA, XPC and XPD). Our results show that lentiviruses are efficient tools to transduce XP fibroblasts and correct repair-defective cellular phenotypes by recovering proper gene expression, normal UV survival and unscheduled DNA synthesis after UV radiation. We propose lentiviral vectors as an attractive alternative for gene therapy protocols focusing on DNA repair genetic diseases.     

The recent transfer of a homing endonuclease gene

The myxomycete Didymium iridis (isolate Panama 2) contains a mobile group I intron named Dir.S956-1 after position 956 in the nuclear small subunit (SSU) rRNA gene. The intron is efficiently spread through homing by the intron-encoded homing endonuclease I-DirI. Homing endonuclease genes (HEGs) usually spread with their associated introns as a unit, but infrequently also spread independent of introns (or inteins). Clear examples of HEG mobility are however sparse. Here, we provide evidence for the transfer of a HEG into a group I intron named Dir.S956-2 that is inserted into the SSU rDNA of the Costa Rica 8 isolate of D.iridis. Similarities between intron sequences that flank the HEG and rDNA sequences that flank the intron (the homing endonuclease recognition sequence) suggest that the HEG invaded the intron during the recent evolution in a homing-like event. Dir.S956-2 is inserted into the same SSU site as Dir.S956-1. Remarkably, the two group I introns encode distantly related splicing ribozymes with phylogenetically related HEGs inserted on the opposite strands of different peripheral loop regions. The HEGs are both interrupted by small spliceosomal introns that must be removed during RNA maturation.     

The spread of LAGLIDADG homing endonuclease genes in rDNA

Group I introns that encode homing endonuclease genes (HEGs) are highly invasive genetic elements. Their movement into a homologous position in an intron-less allele is termed homing. Although the mechanism of homing is well understood, the evolutionary relationship between HEGs and their intron partners remains unclear. Here we have focused on the largest family of HEGs (encoding the protein motif, LAGLIDADG) to understand how HEGs and introns move in rDNA. Our analysis shows the phylogenetic clustering of HEGs that encode a single copy of the LAGLIDADG motif in neighboring, but often evolutionarily distantly related, group I introns. These endonucleases appear to have inserted into existing introns independent of ribozymes. In contrast, our data support a common evolutionary history for a large family of heterologous introns that encode HEGs with a duplicated LAGLIDADG motif. This finding suggests that intron/double-motif HEG elements can move into heterologous sites as a unit. Our data also suggest that a subset of the double-motif HEGs in rDNA originated from the duplication and fusion of a single-motif HEG encoded by present-day ribozymes in LSU rDNA.     

Distribution, sequence homology, and homing of group I introns among T-even-like bacteriophages: evidence for recent transfer of old introns

Self-splicing group I introns are being found in an increasing number of bacteriophages. Most introns contain an open reading frame coding for a homing endo-nuclease that confers mobility to both the intron and the homing endonuclease gene (HEG). The frequent occurrence of intron/HEG has raised questions whether group I introns are spread via horizontal transfer between phage populations. We have determined complete sequences for the known group I introns among T-even-like bacteriophages together with sequences of the intron-containing genes td, nrdB, and nrdD from phages with and without introns. A previously uncharacterized phage isolate, U5, is shown to contain all three introns, the only phage besides T4 found with a "full set" of these introns. Sequence analysis of td and nrdB genes from intron-containing and intronless phages provides evidence that recent horizontal transmission of introns has occurred among the phages. The fact that several of the HEGs have suffered deletions rendering them non-functional implies that the homing endonucleases are of no selective advantage to the phage and are rapidly degenerating and probably dependent upon frequent horizontal transmissions for maintenance within the phage populations. Several of the introns can home to closely related intronless phages during mixed infections. However, the efficiency of homing varies and is dependent on homology in regions flanking the intron insertion site. The occurrence of optional genes flanking the respective intron-containing gene can strongly affect the efficiency of homing. These findings give further insight into the mechanisms of propagation and evolution of group I introns among the T-even-like bacteriophages.     

Adaptation for horizontal transfer in a homing endonuclease

Selfish genes of no function other than self-propagation are susceptible to degeneration if they become fixed in a population, and regular transfer to new species may be the only means for their long-term persistence. To test this idea we surveyed 24 species of yeast for VDE, a nuclear, intein-associated homing endonuclease gene (HEG) originally discovered in Saccharomyces cerevisiae. Phylogenetic analyses show that horizontal transmission has been a regular occurrence in its evolutionary history. Moreover, VDE appears to be specifically adapted for horizontal transmission. Its 31-bp recognition sequence is an unusually well-conserved region in an unusually well-conserved gene. In addition, the nine nucleotide sites most critical for homing are also unusually well conserved. Such adaptation for horizontal transmission presumably arose as a consequence of selection, both among HEGs at different locations in the genome and among variants at the same location. The frequency of horizontal transmission must therefore be a key feature constraining the distribution and abundance of these genes.     

Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA

RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes.     

Insertion of a reversible redox switch into a rare-cutting DNA endonuclease

Target sites for homing endonucleases occur infrequently in complex genomes. As a consequence, these enzymes can be used in mammalian systems to introduce double-strand breaks at recognition sites inserted within defined loci to study DNA repair by homologous and nonhomologous recombination. Using homing endonucleases for gene targeting in vivo would be more feasible if temporal or spatial regulation of their enzymatic activity were possible. Here, we show that the DNA cleavage activity of the yeast PI-SceI homing endonuclease can be turned on and off using a redox switch. Two cysteine pairs (Cys-64/Cys-344 and Cys-67/Cys-365) were separately inserted into flexible DNA binding loop(s) to create disulfide bonds that lock the endonuclease into a nonproductive conformation. The cleavage activities of the reduced Cys-64/Cys-344 and Cys-67/Cys-365 variants are similar or slightly lower than that of the control protein, but the activities of the proteins in the oxidized state are decreased more than 30-fold. Modulating the activity of the proteins is easily accomplished by adding or removing the reducing agent. We show that defects in DNA binding account for the decreased DNA cleavage activities of the proteins containing disulfide bonds. Interestingly, the Cys-67/Cys-365 variant toggles between two different DNA binding conformations under reducing and oxidizing conditions, which may permit the identification of structural differences between the two states. These studies demonstrate that homing endonuclease activity can be controlled using a molecular switch.     

A genetic screen for DNA double-strand break repair mutations in Drosophila

The study of DNA double-strand break (DSB) repair has been greatly facilitated by the use of rare-cutting endonucleases, which induce a break precisely at their cut sites that can be strategically placed in the genome. We previously established such a system in Drosophila and showed that the yeast I-SceI enzyme cuts efficiently in Drosophila cells and those breaks are effectively repaired by conserved mechanisms. In this study, we determined the genetic requirements for the repair of this I-SceI-induced DSB in the germline. We show that Drosophila Rad51 and Rad54 are both required for homologous repair by gene conversion, but are dispensable for single-strand annealing repair. We provided evidence suggesting that Rad51 is more stringently required than Rad54 for intersister gene conversion. We uncovered a significant role of DNA ligase IV in nonhomologous end joining. We conducted a screen for candidate mutations affecting DSB repair and discovered novel mutations in genes that include mutagen sensitive 206, single-strand annealing reducer, and others. In addition, we demonstrated an intricate balance among different repair pathways in which the cell differentially utilizes repair mechanisms in response to both changes in the genomic environment surrounding the break and deficiencies in one or the other repair pathways.     

Invasion of a multitude of genetic niches by mobile endonuclease genes

Persistence of a mobile DNA element in a population reflects a balance between the ability of the host to eliminate the element and the ability of the element to survive and to disseminate to other individuals. In each of the three biological kingdoms, several families of a mobile DNA element have been identified which encode a single protein that acts on nucleic acids. Collectively termed homing endonuclease genes (HEGs), these elements employ varied strategies to ensure their survival. Some members of the HEG families have a minimal impact on host fitness because they associate with genes having self-splicing introns or inteins that remove the HEGs at the RNA or protein level. The HEG and the intron/intein gene spread throughout the population by a gene conversion process initiated by the HEG-encoded endonuclease called 'homing' in which the HEG and intron/intein genes are copied to cognate alleles that lack them. The endonuclease activity also contributes to a high frequency of lateral transmission of HEGs between species as has been documented in plants and other systems. Other HEGs have positive selection value because the proteins have evolved activities that benefit their host organisms. The success of HEGs in colonizing diverse genetic niches results from the flexibility of the encoded endonucleases in adopting new specificities.     

Isolation and characterization of new homing endonuclease specificities at individual target site positions

Homing endonucleases are highly specific DNA endonucleases, encoded within mobile introns or inteins, that induce targeted recombination, double-strand repair and gene conversion of their cognate target sites. Due to their biological function and high level of target specificity, these enzymes are under intense investigation as tools for gene targeting. These studies require that naturally occurring enzymes be redesigned to recognize novel target sites. Here, we report studies in which the homodimeric LAGLIDADG homing endonuclease I-CreI is altered at individual side-chains corresponding to contact points to distinct base-pairs in its target site. The resulting enzyme constructs drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro. Crystal structures of two of these constructs demonstrate that substitution of individual side-chain/DNA contact patterns can occur with almost no structural deformation or rearrangement of the surrounding complex, facilitating an isolated, modular redesign strategy for homing endonuclease activity and specificity.     

A modular toolset for recombination transgenesis and neurogenetic analysis of Drosophila

Transgenic Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues.     

A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA

DiGIR1 is a group I-like cleavage ribozyme found as a structural domain within a nuclear twin-ribozyme group I intron. DiGIR1 catalyzes cleavage by branching at an Internal Processing Site (IPS) leading to formation of a lariat cap at the 5′-end of the 3′-cleavage product. The 3′-cleavage product is subsequently processed into an mRNA encoding a homing endonuclease. By analysis of combinations of 5′- and 3′-deletions, we identify a hairpin in the 5′-UTR of the mRNA (HEG P1) that is formed by conformational switching following cleavage. The formation of HEG P1 inhibits the reversal of the branching reaction, thus giving it directionality. Furthermore, the release of the mRNA is a consequence of branching rather than hydrolytic cleavage. A model is put forward that explains the release of the I-DirI mRNA with a lariat cap and a structured 5′-UTR as a direct consequence of the DiGIR1 branching reaction. The role of HEG P1 in GIR1 branching is reminiscent of that of hairpin P-1 in splicing of the Tetrahymena rRNA group I intron and illustrates a general principle in RNA-directed RNA processing.     

Homing endonuclease genes: the rise and fall and rise again of a selfish element

Homing endonuclease genes (HEGs) are selfish genetic elements that spread by first cleaving chromosomes that do not contain them and then getting copied across to the broken chromosome as a byproduct of the repair process. The success of this strategy will depend on the opportunities for homing--in other words, the frequency with which HEG(+) and HEG(-) chromosomes come into contact--which varies widely among host taxa. HEGs are also unusual in that the selection pressure for endonuclease function disappears if they become fixed in a population, which makes them susceptible to degeneration and imposes a need for regular horizontal transmission between species. HEGs will be selected to reduce the harm done to the host organism, and this is expected to influence the evolution of their sequence specificity and maturase functions. HEGs may also be domesticated by their hosts, and are currently being put to human uses.     

Generation of highly site-specific DNA double-strand breaks in human cells by the homing endonucleases I-PpoI and I-CreI

We have determined the ability of two well-characterized eukaryotic homing endonucleases, I-PpoI from the myxomycete Physarum polycephalum and I-CreI from the green alga Chlamydomonas reinhardtii, to generate site-specific DNA double-strand breaks in human cells. These 18-kDa proteins cleave highly conserved 15- or 24-bp rDNA homing sites in their respective hosts to generate homogeneous 4-base, 3' ends that initiate target intron transposition or "homing." We show that both endonucleases can be expressed in human cells and can generate site-specific DNA double-strand breaks in 28S rDNA and homing site plasmids. These endonuclease-induced breaks can be repaired in vivo, although break repair is mutagenic with the frequent generation of short deletions or insertions. I-PpoI and I-CreI should be useful for analyzing DNA double-strand break repair in human cells and rDNA.     

Gamma-retroviral vectors enveloped with an antibody and an engineered fusogenic protein achieved antigen-specific targeting

Development of methods to engineer gamma-retroviral vectors capable of transducing target cells in a cell-specific manner could impact the future of the clinical application of gene therapy as well as the understanding of the biology of transfer gene vectors. Two molecular events are critical for controlling the entry of gamma-retroviral vectors to target cells: binding to cell-surface receptors and the subsequent fusion of viral vector membrane and cellular membrane. In this report, we evaluated a method to incorporate a membrane-bound antibody and a fusogenic molecule to provide binding and fusion functions respectively, into gamma-retroviral vectors for targeted gene delivery. An anti-CD20 antibody and a fusogenic protein derived from Sindbis virus glycoprotein could be efficiently co-displayed on the surface of viral vectors. Vectors bearing anti-CD20 antibody conferred their binding specificity to cells expressing CD20. Enhanced in vitro transduction towards CD20-expressing cells was observed for gamma-retroviral vectors displaying both an antibody and a fusogen. We found that the biological activity of the fusogen played an important role on the efficiency of such a targeting strategy and were able to engineer several mutant forms of the fusogen exhibiting elevated fusion function to improve the overall efficiency of targeted transduction. We devised an animal model to show that subcutaneous injection of such engineered vectors to the areas xenografted with target cells could achieve targeted gene delivery in vivo. Taken together, we demonstrated as proof-of-principle a flexible and modular two-molecule strategy for engineering targeting gamma-retroviral vectors.