Native homing endonucleases can target conserved genes in humans and in animal models

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assay

Evidence from phylogenetic and genome fingerprinting analyses suggests rapidly changing variation in Halorubrum and Haloarcula populations

Halobacteria require high NaCl concentrations for growth and are the dominant inhabitants of hypersaline environments above 15% NaCl. They are well documented to be highly recombinogenic, both in frequency and in the range of exchange partners. In this study, we examine the genetic and genomic variation of cultured, naturally co-occurring environmental populations of Halobacteria. Sequence data from multiple loci (~2500bp) identified closely related strains belonging to the genera Halorubrum and Haloarcula. Genome fingerprinting using a random priming PCR amplification method to analyze these isolates revealed diverse banding patterns within and across each of the genera and surprisingly even for isolates that are identical at the nucleotide level for five protein coding sequenced loci. This variance in genome structure even between identical multilocus sequence analysis (MLSA) haplotypes suggests that accumulation of variation is rapid, perhaps occurring every generation

In Vivo Characterization of the Homing Endonuclease within the polB Gene in the Halophilic Archaeon Haloferax volcanii

Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN) gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB) contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type

Native homing endonucleases can target conserved genes in humans and in animal models

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays

Molecular Biology of Archaea II Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions

Abstract Inteins are selfish genetic elements that disrupt the sequence of protein-coding genes and are excised post-translationally. Most inteins also contain a HEN (homing endonuclease) domain, which is important for their horizontal transmission. The present review focuses on the evolution of inteins and their nested HENs, and highlights several unsolved questions that could benefit from molecular genetic approaches. Such approaches can be well carried out in halophilic archaea, which are naturally intein-rich and have highly developed genetic tools for their study. In particular, the fitness effects of habouring an intein/HEN can be tested in direct competition assays, providing additional insights that will improve current evolutionary models. HENs (homing endonucleases) HENs are a large and diverse class of selfish elements found in archaea, bacteria and lower eukaryotes, and their respective viruses. HENs recognize and cleave specifically long target sequences (12-40 bp) that typically occur only once in a given genome Mutualism between selfish elements Although it is obvious that inteins can profit from horizontal invasion, how and why the HEN benefits from its association with the intein is less obvious. Since HENs typically confer no selective advantage upon their host organism, they can only persist as long as the rate of their dissemination surpasses the rate of their degeneration via genetic drift or counterselection. The rate of dissemination is in turn dependent on the availability of homing targets, which are also subjects of mutation and selection. If cleavage by the HEN is by any degree toxic to the host, mutations in the target site may be selected that prevent it from being recognized or cleaved

Inclusion of Ultra-Orthodox Students in Higher Education: A Case Study about Women Seminary in the Engineering College of Jerusalem

This study examines how far the establishment of an ultra-orthodox (Haredi) women-only curriculum and learning environment crafted for their needs, such as supplementary Biblical studies, fosters students’ enrollment and achievement in STEM. The methodology utilizes interviews with administrators accompanied by a Google questionnaire surveying students in order to compare the secular Azrieli College of Engineering with its new ultra-orthodox branch, Tmura Seminary, which aims to promote the acquisition by ultra-orthodox Jewish women of an engineering diploma so that they can find employment in the job market. This objective is significant because the ultra-orthodox community in Israel represents a unique family setting in which the men devote most of their workday to Judaic studies while the women are the breadwinners. The findings of the current study show that addressing the higher education gender gap by purposeful efforts to include ultra-orthodox women is indeed feasible. Still, it remains a challenging endeavor because besides being first-generation students subject to religious constraints, Haredi students are insufficiently prepared in mathematics and English before arriving at the campus, which requires that they participate in an intensive pre-academic preparatory program. The analysis of results showed differences between ultra-orthodox and secular students in their motivation for study, sources of information about suitable education institutions, balancing work and family life priorities, financial need for scholarships, and career path, which is directed toward finding a job in the high-tech industry rather than continuing after graduation to pursue a Master-level degree

Inclusion of Ultra-Orthodox Students in Higher Education: A Case Study about Women Seminary in the Engineering College of Jerusalem

This study examines how far the establishment of an ultra-orthodox (Haredi) women-only curriculum and learning environment crafted for their needs, such as supplementary Biblical studies, fosters students’ enrollment and achievement in STEM. The methodology utilizes interviews with administrators accompanied by a Google questionnaire surveying students in order to compare the secular Azrieli College of Engineering with its new ultra-orthodox branch, Tmura Seminary, which aims to promote the acquisition by ultra-orthodox Jewish women of an engineering diploma so that they can find employment in the job market. This objective is significant because the ultra-orthodox community in Israel represents a unique family setting in which the men devote most of their workday to Judaic studies while the women are the breadwinners. The findings of the current study show that addressing the higher education gender gap by purposeful efforts to include ultra-orthodox women is indeed feasible. Still, it remains a challenging endeavor because besides being first-generation students subject to religious constraints, Haredi students are insufficiently prepared in mathematics and English before arriving at the campus, which requires that they participate in an intensive pre-academic preparatory program. The analysis of results showed differences between ultra-orthodox and secular students in their motivation for study, sources of information about suitable education institutions, balancing work and family life priorities, financial need for scholarships, and career path, which is directed toward finding a job in the high-tech industry rather than continuing after graduation to pursue a Master-level degree

Toxoplasma Controls Host Cyclin E Expression through the Use of a Novel MYR1-Dependent Effector Protein, HCE1

Like most Apicomplexan parasites, Toxoplasma gondii has the remarkable ability to invade and establish a replicative niche within another eukaryotic cell, in this case, any of a large number of cell types in almost any warm-blooded animals. Part of the process of establishing this niche is the export of effector proteins to co-opt host cell functions in favor of the parasite. Here we identify a novel effector protein, HCE1, that the parasites export into the nucleus of human cells, where it modulates the expression of multiple genes, including the gene encoding cyclin E, one of the most crucial proteins involved in controlling when and whether a human cell divides. We show that HCE1 works through binding to specific transcription factors, namely, E2F3, E2F4, and DP1, that normally carefully regulate these all-important pathways. This represents a new way in which these consummately efficient infectious agents co-opt the human cells that they so efficiently grow within.Toxoplasma gondii is an obligate intracellular parasite that establishes a favorable environment in the host cells in which it replicates. We have previously reported that it uses MYR-dependent translocation of dense granule proteins to elicit a key set of host responses related to the cell cycle, specifically, E2F transcription factor targets, including cyclin E. We report here the identification of a novel Toxoplasma effector protein that is exported from the parasitophorous vacuole in a MYR1-dependent manner and localizes to the host’s nucleus. Parasites lacking this inducer of host cyclin E (HCE1) are unable to modulate E2F transcription factor target genes and exhibit a substantial growth defect. Immunoprecipitation of HCE1 from infected host cells showed that HCE1 efficiently binds elements of the cyclin E regulatory complex, namely, DP1 and its partners E2F3 and E2F4. Expression of HCE1 in Neospora caninum, or in uninfected human foreskin fibroblasts (HFFs), showed localization of the expressed protein to the host nuclei and strong cyclin E upregulation. Thus, HCE1 is a novel effector protein that is necessary and sufficient to impact the E2F axis of transcription, resulting in co-opting of host functions to the advantage of Toxoplasma

MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects

The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma, we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔmyr1, and RHΔasp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔmyr1- but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions.IMPORTANCEToxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins