Topic
Homing endonuclease
About: Homing endonuclease is a research topic. Over the lifetime, 759 publications have been published within this topic receiving 36112 citations.
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TL;DR: A procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences is described.
Abstract: Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however, for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here we describe a procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring an infectious herpesvirus clone to demonstrate the potential of the mutagenesis technique, which was used for the insertion of long sequences such as coding regions or promoters, introduction of point mutations, scarless deletions, and insertion of short sequences such as an epitope tag. The system proved to be highly reliable and efficient and can be adapted for a variety of different modifications of BAC clones, which are fundamental tools for applications as diverse as the generation of transgenic animals and the construction of gene therapy or vaccine vectors.
738 citations
New England Biolabs1, New York State Department of Health2, Columbia University3, Wayne State University4, University of Basel5, University of Toledo6, University of Edinburgh7, University of Alabama8, University of Portsmouth9, Moscow State University10, University of Illinois at Chicago11, University of Bristol12, University of Rochester13, Duke University14, University of Sheffield15, Vilnius University16, University of Giessen17, University of Copenhagen18, Hungarian Academy of Sciences19, North Carolina State University20, University of Tokyo21, Humboldt University of Berlin22, Brookhaven National Laboratory23, University of Massachusetts Medical School24, National Institutes of Health25, Indian Institute of Science26, University of Warsaw27, University of California, Santa Barbara28, State Research Center of Virology and Biotechnology VECTOR29, University of Oregon30, The Chinese University of Hong Kong31, University of Maryland, College Park32, Fred Hutchinson Cancer Research Center33, University of Wisconsin-Madison34, University of Nebraska–Lincoln35, University of Lisbon36
TL;DR: In this article, a nomenclature for restriction endonucleases, DNA methyltransferases, homing endon nucleases and related genes and gene products is described.
Abstract: A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.
710 citations
TL;DR: It is shown that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants and the Cas9 nickase has the potential to become an important tool for genome engineering in plants.
Abstract: Engineered nucleases can be used to induce site-specific double-strand breaks (DSBs) in plant genomes Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error-prone non-homologous end-joining (NHEJ) Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ Cas9 can also be engineered to work as a nickase inducing single-strand breaks (SSBs) Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants We demonstrate the stable inheritance of nuclease-induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 25 up to 700% Deep sequencing analysis revealed NHEJ-mediated DSB repair in about a third of all reads in T1 plants In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740-fold Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I-SceI Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB-inducing enzymes Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants It should not only be applicable for HR-mediated gene targeting systems but also by the combined action of two nickases as DSB-inducing agents excluding off-target effects in homologous genomic regions
622 citations
TL;DR: The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.
Abstract: Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.
588 citations
TL;DR: The evolution of homing endonucleases is considered, both at the structure-function level and in terms of their persistence in widely divergent biological systems.
Abstract: Homing endonucleases are rare-cutting enzymes encoded by introns and inteins. They have striking structural and functional properties that distinguish them from restriction enzymes. Nomenclature conventions analogous to those for restriction enzymes have been developed for the homing endonucleases. Recent progress in understanding the structure and function of the four families of homing enzymes is reviewed. Of particular interest are the first reported structures of homing endonucleases of the LAGLIDADG family. The exploitation of the homing enzymes in genome analysis and recombination research is also summarized. Finally, the evolution of homing endonucleases is considered, both at the structure-function level and in terms of their persistence in widely divergent biological systems.
539 citations