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

Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions

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.</jats:p

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&rsquo; 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