45 results about "Homing endonuclease" patented technology

A group of modular cloning vector plasmids for the synthesis of a transgene or other complicated DNA construct, by providing a backbone having docking points therein, for the purpose of gene expression or analysis of gene expression. The invention is useful for assembling a variety of DNA fragments into a de novo DNA construct or transgene by using cloning vectors optimized to reduce the amount of manipulation frequently needed. The module vector contains at least one multiple cloning site (MCS) and multiple sets of rare restriction and/or unique homing endonuclease (“HE”) sites, arranged in a linear pattern. This arrangement defines a modular architecture that allows the user to place domain modules or inserts into a PE3 transgene vector construct without disturbing the integrity of DNA elements already incorporated into the PE3 vector in previous cloning steps. The PE3 transgenes produced using the invention may be used in a single organism, or in a variety of organisms including bacteria, yeast, mice, and other eukaryotes with little or no further modification.

Compositions and methods for modifying genetic material are provided. One embodiment provides aptamers capable of binding to a site-specific DNA binding moiety to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting or AGT). One embodiment provides an oligonucleotide containing a aptamer, preferably a DNA aptamer at the 5′ end. The oligonucleotide also contains a region of homology, also referred to as donor DNA, to a desired nucleic acid, locus, or gene. The DNA binding moiety can be a nucleic acid, a protein, or a complex of proteins. In a preferred embodiment the DNA binding moiety is a homing endonuclease that cuts DNA to facilitate the modification of the DNA by the donor DNA.

An engineered highly specific DNA-cleavage enzyme delivers a site-specific nick in a double stranded DNA, to cleave one DNA strand within its target site while leaving the opposing DNA strand intact. The engineered enzyme provides the ability to induce a gene conversion event in a mammalian cell. An engineered sequence-specific nickase derived from a LAGLIDADG homing endonuclease is altered by a single amino acid residue, wherein the amino acid residue is involved in the polarization of solvent molecules and acid-base catalysis in the active site without affecting direct contacts between the enzyme and either the bound DNA or bound metal ions. Engineered, site-specific nickase variants, such as of I-AniI and other homing endonucleases, are particularly useful in targeted genome engineering as well as therapeutic, targeted gene repair.

The invention discloses controllable vector elimination method as well as an easy-to-use CRISPR-Cas9 tool. The invention provides a controllable vector elimination tool, which is composed of a control module and an elimination module, the control module including: a core zone which contains an elaborately-controlling promoter and a homing endonuclease encoding gene, two terminators respectively at two ends of the core zone, and homing endonuclease recognition sequences on the outer ends of the two terminators; and the elimination module including: an antibiotic resistance gene expression cassette, and homing endonuclease recognition sequences on the two ends; the tool can easily, quickly and high-effectively remove a plasmid vector in one step. A further developed easy-to-use CRISPR-Cas9 system can achieve one-step quick elimination to Cas9 and sgRNA encoding plasmids from a host cell. The easy-to-use CRISPR-Cas9 system is very convenient and practical, greatly saves experiment time, reduces experiment intensity, and is a very practical gene editing tool.

The present invention provides in vivo systems in which activity of a biological cleavage enzyme, such as a site-specific recombinase, a homing endonuclease, or an intein, is linked to cell viability and therefore can be selected. The invention further provides methods of making cells in which the activity of a biological cleavage enzyme is linked to viability, as well as methods of identifying new biological cleavage enzymes, including enzymes having altered site specificity, using such cells.

Disclosed herein are compositions for inactivating the human TCR-alpha gene comprising engineered LAGLIDADG homing endonucleases (LHEs) and their derivatives, particularly derived from members of the \-Onul subfamily of LHEs. Polynucleotides encoding such endonucleases, vectors comprising said polynucleotides, cells comprising or having been treated with such endonucleases, and therapeutic compositions deriving therefore are also provided.

The present invention provides in vivo systems in which activity of a biological cleavage enzyme, such as a site-specific recombinase, a homing endonuclease, or an intein, is linked to cell viability and therefore can be selected. The invention further provides methods of making cells in which the activity of a biological cleavage enzyme is linked to viability, as well as methods of identifying new biological cleavage enzymes, including enzymes having altered site specificity, using such cells.

The present disclosure provides improved genome editing compositions and methods for editing a BCL11A gene. The disclosure further provides genome edited cells for the prevention, treatment, or amelioration of at least one symptom of a hemoglobinopathy.

Nucleic acids, compositions, and methods that allow for the excision of one or more loci from the genome of a host cell are provided herein. In particular, provided herein is an excisable nucleic acid construct comprising, in a 5' to 3' orientation: a first tandem repeat nucleic acid, a first homing endonuclease recognition site, a target nucleic acid, a second homing endonuclease recognition site, and a second tandem repeat nucleic acid. In some embodiments, the excisable nucleic acid construct is integrated into the host cell genome, and the target nucleic acid can be excised from the host cell genome by contacting the homing endonuclease recognition sites with one or more appropriate homing endonucleases.

The invention belongs to the technical field of genetic engineering and relates to a 2-methyl citric acid high-yield genetic engineering bacterium and a construction method thereof. The 2-methyl citric acid high-yield genetic engineering bacterium is obtained by knocking out a 2-methyl citric acid dehydrase gene prpD in a bacillus thuringiensis BMB171 genome. According to the construction method of the 2-methyl citric acid high-yield genetic engineering bacterium, bacillus thuringiensis BMB171 is adopted as an original strain to successfully obtain a 2-methyl citric acid dehydrase gene prpD traceless deletion mutant strain delta prpD by the aid of a homing endonuclease I-SceI induced chromosome homologous recombination mechanism based gene knockout system. According to liquid chromatography and mass spectrometry detection, the intracellular 2-methyl citric acid concentration of delta prpD strain is increased by 217 times as compared with that of the original strain BMB171, and the 2-methyl citric acid prepared according to the method is specific in steric configuration as compared with 2-methyl citric acid synthesized according to a chemical method.

According to particular exemplary aspects, DNA target site binding and cleavage properties of native, variant or modified homing endonucleases (HE) (e.g., LAGLIDAG (LHE), HNH, His-Cys Box, GIY-YIG, I-SspI-type, and fusions, muteins or variants thereof) in solution are recapitulated on the cell surface (e.g., as assessed by flow cytometric analysis) to provide for novel cells expressing one or more cell surface HEs (e.g., expressing one or more HE binding and/or cleavage specificities), novel cell libraries, and high-throughput methods for assessing target site binding, target site cleavage. The rapid analysis of HE and LHE-DNA interactions on the cell surface with concurrent sorting options provides for high-throughput library screening affording rapid identification, analysis and isolation of novel HEs or LHEs having novel sequence specificities. Such novel sequence specificities, obtained by said methods provide novel methods for introducing targeted DNA-strand cleavage events, and novel chromatin immunoprecipitation methods (CHIP methods).

The invention provides improved genome editing compositions and methods for editing a TIM3 gene. The invention further provides genome edited cells for the prevention, treatment, or amelioration of at least one symptom of, a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.

The invention provides improved genome editing compositions and methods for editing an IL-10Rα gene. The invention further provides genome edited cells for the prevention, treatment, or amelioration of at least one symptom of, a cancer, GVHD, a transplant rejection, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.

The invention provides pharmaceutical composition having as an active ingredient either a homing endonuclease (HE) capable of cleaving a non-native target nucleotide sequence in a genome or a nucleotide sequence encoding for a HE capable of cleaving a target site in a non-native genome. The invention also provides uses for such HEs, and methods of treatment utilizing such HEs. The HE may be, for example, any one of the HEs PI-SceI, POLB HE, PRP8 HE, or Nostoc species PCC7120 HE.

The present disclosure provides improved genome editing compositions and methods for editing a PD-1 gene. The disclosure further provides genome edited cells for the prevention, treatment, or amelioration of at least one symptom of, a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.