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Toolgen Incorporated and Institute For Basic Science | Date: 2017-04-05

The present invention relates to a method for analyzing a genotype using a target-specific nuclease and, specifically, to a method for diagnosing cancer or analyzing a genotype by removing wild type DNA or particular genotype DNA using a target-specific nuclease or a variant thereof to amplify or concentrate only a small amount of DNA which has a difference in variation, such as a mutation, or genotype, and to a method for separating target DNA sesusing a target-specific nuclease or a variant thereof. Such methods are novel paradigm methods contrary to existing simple target-specific nucleases for post-PCR recognition of normal genotype and carcinogenic genotype, and can be favorably used in the early diagnosis of cancer or analysis of similar genotypes.

SEOUL, South Korea, Feb. 22, 2017 (GLOBE NEWSWIRE) -- ToolGen, Inc. (KONEX, 199800), a biotechnology company specializing in genome editing, today announced encouraging data from a study evaluating Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 orthologue characterized to date, for efficient genome editing in vivo. Results from the study entitled “In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni,” were published online in the peer-reviewed journal Nature Communications. The study was led by researchers from ToolGen, Dr. Jin-soo Kim, Principal Investigator, from the Institute for Basic Science and Dr. Jeong Hun Kim from Seoul National University Hospital. The study demonstrated the ability to package the CjCas9 gene, its sgRNA sequence into a single Adeno-associated virus (AAV) vector for in vivo gene surgery. Furthermore, CjCas9 was shown to be highly specific in cleaving the target sites in the human or mouse genome in vitro, which could have significant potential precision genome editing and gene surgery. CjCas9 delivered via AAV, induced target mutations in mouse muscle cells and retinal pigment epithelium cells (RPE) with no off-target mutations detected in the genome. The study confirmed that CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduced the size of laser-induced choroidal neovascularization, a condition leading to the formation of new blood vessels in the choroid layer of the eye. This suggests that gene surgery with CjCas9 could be a promising treatment option for age-related macular degeneration, a leading cause of blindness in adults. Seokjoong Kim, Research Director of ToolGen commented, “Despite the recent advances in CRISPR/Cas9 genome editing technology, it has been difficult to package a whole cassette of Cas9 and sgRNA into certain viral vectors such as AAV owing to the large size of the most commonly used Cas9 genes. The need to split the gene and package it into multiple AAV vectors results in a less than optimal delivery method, and the split gene is less active than its intact counterpart. However, our study, utilizing CjCas9, which is significantly smaller than other commonly used Cas9 genes, enables us to overcome these problems. We are very pleased with the findings obtained from this study and strongly believe that this data can serve as a foundation for further exploration in the CRISPR/Cas9 arena.” Jeong Hun Kim, Clinical Professor in the Department of Ophthalmology, Seoul National University Hospital commented, “This work shows promising results for the application of CRISPR/Cas9 for eye diseases. We look forward to extending these observations on the successful modulation of neovascularization through targeted in vivo genome editing to additional disease models and large animals that could potentially establish a new class of therapeutic options. Additionally, with an efficient genome editing process established in the eye, we can broaden target indications to include rare diseases.” For more information, contact Seokjoong Kim, Director of Research Center Tel. +8210-6776-7824, ToolGen, Inc. is a biotechnology company focused on the development and application of genome editing technologies. It creates, and holds intellectual property rights for essential tools and technologies for editing the genetic information in microbial, plant, animal, and human cells. ToolGen’s mission is to translate the potential of our innovative platform technology into transformative products for biomedicine and agriculture. For more information, please visit

Kim D.,Korea Basic Science Institute | Kim D.,Seoul National University | Bae S.,Korea Basic Science Institute | Bae S.,Seoul National University | And 9 more authors.
Nature Methods | Year: 2015

Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5â €2 ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9. © 2015 Nature America, Inc.

Lee H.J.,Seoul National University | Lee H.J.,Washington University in St. Louis | Kweon J.,Seoul National University | Kim E.,Seoul National University | And 3 more authors.
Genome Research | Year: 2012

Despite the recent discoveries of and interest in numerous structural variations (SVs) - which include duplications and inversions - in the human and other higher eukaryotic genomes, little is known about the etiology and biology of these SVs, partly due to the lack of molecular tools with which to create individual SVs in cultured cells and model organisms. Here, we present a novelmethod of inducing duplications and inversions in a targeted manner without pre-manipulation of the genome. We found that zinc finger nucleases (ZFNs) designed to target two different sites in a human chromosome could introduce two concurrent double-strand breaks, whose repair via non-homologous end-joining (NHEJ) gives rise to targeted duplications and inversions of the genomic segments of up to a mega base pair (bp) in length between the two sites. Furthermore, we demonstrated that a ZFN pair could induce the inversion of a 140-kbp chromosomal segment that contains a portion of the blood coagulation factor VIII gene to mimic the inversion genotype that is associated with some cases of severe hemophilia A. This same ZFN pair could be used, in theory, to revert the inverted region to restore genomic integrity in these hemophilia A patients.We propose that ZFNs can be employed asmolecular tools to study mechanisms of chromosomal rearrangements and to create SVs in a predetermined manner so as to study their biological roles. In addition, our method raises the possibility of correcting genetic defects caused by chromosomal rearrangements and holds new promise in gene and cell therapy. © 2012 by Cold Spring Harbor Laboratory Press.

Kim J.M.,Seoul National University | Kim D.,Seoul National University | Kim S.,ToolGen Inc. | Kim J.-S.,Seoul National University
Nature Communications | Year: 2014

Restriction fragment length polymorphism (RFLP) analysis is one of the oldest, most convenient and least expensive methods of genotyping, but is limited by the availability of restriction endonuclease sites. Here we present a novel method of employing CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) in RFLP analysis. We prepare RGENs by complexing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed guide RNAs that are complementary to the DNA sequences of interest. Then, we genotype recurrent mutations found in cancer and small insertions or deletions (indels) induced in cultured cells and animals by RGENs and other engineered nucleases such as transcription activator-like effector nucleases (TALENs). Unlike T7 endonuclease I or Surveyor assays that are widely used for genotyping engineered nuclease-induced mutations, RGEN-mediated RFLP analysis can detect homozygous mutant clones that contain identical biallelic indel sequences and is not limited by sequence polymorphisms near the nuclease target sites. © 2014 Macmillan Publishers Limited. All rights reserved.

Sung Y.H.,Yonsei University | Jin Y.,Yonsei University | Kim S.,ToolGen Inc. | Lee H.-W.,Yonsei University
Methods | Year: 2014

The use of engineered nucleases in one-cell stage mouse embryos is emerging as an efficient alternative to conventional gene targeting in mouse embryonic stem (ES) cells. These nucleases are designed or reprogrammed to specifically induce double strand breaks (DSBs) at a desired genomic locus, and efficiently introduce mutations by both error-prone and error-free DNA repair mechanisms. Since these mutations frequently result in the loss or alteration of gene function by inserting, deleting, or substituting nucleotide sequences, engineered nucleases are enabling us to efficiently generate gene knockout and knockin mice. Three kinds of engineered endonucleases have been developed and successfully applied to the generation of mutant mice: zinc-finger nuclease (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs). Based on recent advances, here we provide experimentally validated, detailed guidelines for generating non-homologous end-joining (NHEJ)-mediated mutant mice by microinjecting TALENs and RGENs into the cytoplasm or the pronucleus of one-cell stage mouse embryos. © 2014 Elsevier Inc.

Toolgen Incorporated | Date: 2016-04-06

Pharmaceutical and veterinary preparations for the treatment of hereditary diseases; reagents for use in veterinary genetic testing; reagents for use in medical genetic testing; preparations for detecting mutation in prion genes for medical purposes; stem cells for medical purposes; biotechnological preparations for medical purposes for the treatment of hereditary diseases; cells for medical purposes; diagnostic preparations for medical purposes; cultures of microorganisms for medical purposes; diagnostic preparations for veterinary purposes; biological preparations for medical or veterinary purposes for the treatment of hereditary diseases; surgical implants grown from stem cells; sanitary preparations for medical use. Consultation and providing information in the fields of biomedicine; consultancy in analysis of biology sequence processing for medical research purposes; consultancy in analysis of genomics for medical research purposes; analysis of amino acid for medical purpose; scientific research in the fields of medicine, health and pharmacology; consultation and providing information in the field of pharmacology; structural and functional analysis of genomes; multiple nucleotide sequence analysis; scientific research in the fields of biology; consultation and providing information in the fields of biology; laboratory research in the field of biology; genetic testing for scientific research purposes; gene analysis; drug discovery services; protein analysis; stem cell testing for medical research, treatment, and diagnosis; research and development in the pharmaceutical and biotechnology fields; DNA testing; DNA analysis; DNA sequence analysis; DNA screening for scientific research purposes; research and development of DNA chips; biotechnology research; biotechnology services; biological research; research and development of new products for others; research on the subject of pharmaceuticals; bacteriological research. Medical and health services relating to DNA, genetics and genetic testing; genetic testing for medical purposes; provision of genetic testing for medical treatment purposes; provision of genetic information for medical treatment purposes; provision of gene therapy for medical treatment purposes; genetic counseling for medical purposes; medical diagnostic services; physical examination services; performing diagnosis of diseases; medical services relating to the removal, treatment and processing of stem cells; DNA screening for medical purposes; RNA or DNA analysis for cancer diagnosis and prognosis; analysis of human biodata for medical purposes; medical pathology for the diagnosis and treatment of disease; plant nursery services; animal breeding; consultation and providing information in the fields of medicine and health.

The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.

The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.

The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.

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