Abstract: Disclosed are methods of making and using engineered Fold cleavage domain variants. Also disclosed are methods, compositions and fusion proteins containing obligate heterodimers of engineered Fold cleavage domain variants and DNA binding domains, such as zinc finger protein (ZFP) domains and transcription activator-like effector (TALE) domains.

Abstract: Disclosed are methods of making and using engineered FokI cleavage domain variants. Also disclosed are methods, compositions and fusion proteins containing obligate heterodimers of engineered FokI cleavage domain variants and DNA binding domains, such as zinc finger protein (ZFP) domains and transcription activator-like effector (TALE) domains.

Abstract: ABSTRACT Methods, compositions, constructs, vectors, cell lines, and kits, for generating induced pluripotent stem cells by site-specific integration of pluripotency coding sequences with endonucleases for use in gene therapy, regenerative medicine, cell therapy or drug screening.

Abstract: Disclosed are methods of making and using engineered FokI cleavage domain variants. Also disclosed are methods, compositions and fusion proteins containing obligate heterodimers of engineered FokI cleavage domain variants and DNA binding domains, such as zinc finger protein (ZFP) domains and transcription activator-like effector (TALE) domains.

Abstract: Disclosed herein are chimeric zinc finger endonucleases useful in disrupting and/or replacing at least a portion of a gene of interest (e.g. CFTR, DMPK, CCR5, TYR or ?globin).

Abstract: The invention relates to a novel method for obtaining DNA metasegment comprising ligating adjoining DNA fragments at least 10 Kb in size containing at least one overlapping region.

Abstract: The present invention reveals a method for enzymatically inactivating a target DNA, a method for detecting conformational change in a nucleic acid, and a method for detecting the presence of a target DNA molecule. The method for enzymatically inactivating a target DNA involves preparing a plasmid, phage, virus, or any other delivery vehicle such as a liposome containing a gene encoding a nuclease. The delivery vehicle is then delivered into cells. The cells are induced to produce the nuclease and the target DNA is then enzymatically inactivated. Alternatively, the nuclease protein is delivered directly to cells and used to enzymatically inactivate the target DNA. The method for detecting conformational change in a nucleic acid requires contacting a nucleic acid with a hybrid restriction nuclease, determining whether the hybrid restriction nuclease has interacted with the nucleic acid, and detecting the conformational change in the nucleic acid.

Abstract: The present invention reveals a method for enzymatically inactivating a target DNA, a method for detecting conformational change in a nucleic acid, and a method for detecting the presence of a target DNA molecule. The method for enzymatically inactivating a target DNA involves preparing a plasmid, phage, virus, or any other delivery vehicle such as a liposome containing a gene encoding a nuclease. The delivery vehicle is then delivered into cells. The cells are induced to produce the nuclease and the target DNA is then enzymatically inactivated. Alternatively, the nuclease protein is delivered directly to cells and used to enzymatically inactivate the target DNA. The method for detecting conformational change in a nucleic acid requires contacting a nucleic acid with a hybrid restriction nuclease, determining whether the hybrid restriction nuclease has interacted with the nucleic acid, and detecting the conformational change in the nucleic acid.

Abstract: The present invention reveals methods for cloning hybrid restriction endonucleases and for enzymatically inactivating a target DNA. The method for cloning hybrid restriction endonucleases involves co-expression of a ligase. A first plasmid contains a gene encoding a DNA ligase. A second plasmid contains a gene encoding a hybrid restriction endonuclease and is compatible with the first plasmid. The method involves transfecting host cells with the first plasmid, so that DNA ligase is produced, followed by transfecting the cells with the second plasmid. The method for enzymatically inactivating a target DNA involves preparing a plasmid, phage, virus or any other delivery vehicle such as a liposome containing a gene encoding a nuclease, delivering the gene into cells, inducing the cells to produce the nuclease and enzymatically inactivating the target DNA.

Abstract: The present invention reveals the construction of several insertion (4, 8, 12, 18, 19 or 23 amino acid residues) and deletion (4 or 7 amino acid residues) mutants of the linker region of FokI endonuclease in Flavobacterium okeanokoites. The mutant enzymes were purified, and their cleavage properties were characterized. The mutants have the same DNA sequence-specificity as the wild-type enzyme. However, compared with the wild-type enzyme, the insertion mutants cleave predominantly one nucleotide further away from the recognition site on both strands of the DNA substrate. The four codon deletion mutant shows relaxed specificity at the cut site while the seven codon deletion appears to inactivate the enzyme. The DNA-binding and cleavage domains of FokI appear to be linked by a relatively malleable linker. No simple linear relationship exists between the linker length and the distance of the cut site from the recognition site.

Abstract: The present inventors have identified the recognition and cleavage domains of the FokI restriction endonuclease. Accordingly, the present invention relates to DNA segments encoding the recognition and cleavage domains of the FokI restriction endonuclease, respectively. The 41 kDa N-terminal fragment constitutes the FokI recognition domain while the 25 kDa C-terminal fragment constitutes the FokI cleavage nuclease domain. The present invention also relates to hybrid restriction enzymes comprising the nuclease domain of the FokI restriction endonuclease linked to a recognition domain of another enzyme. One such hybrid restriction enzyme is Ubx-F.sub.N. This enzyme contains the homeo domain of Ubx linked to the cleavage or nuclease domain of FokI. Additionally, the present invention relates to the construction of two insertion mutants of FokI endonuclease.

Abstract: The present inventor have identified of the recognition and cleavage domains of the FokI restriction endonuclease. Accordingly, the present invention relates to DNA segments encoding the recognition and cleavage domains of the FokI restriction endonuclease, respectively. The 41 kDa N-terminal fragment constitutes the FokI recognition domain while the 25 kDa C-terminal fragment constitutes the FokI cleavage nuclease domain. The present invention also relates to hybrid restriction enzymes comprising the nuclease domain of the FokI restriction endonuclease linked to a recognition domain of another enzyme.