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Epigenetically regulated site-specific nucleases

A nuclease, target site technology, applied in the field of site-specific nucleases regulated by epigenetics

Pending Publication Date: 2019-09-27
THE GENERAL HOSPITAL CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, the specificity of the RNA-guided nuclease (RGN) platform of CRISPR-Cas is mainly determined by the guide RNA molecule (guide RNA, gRNA) that is complementary to the target DNA site; other genome editing platforms such as Zinc-finger (ZF) nucleases or TALE nucleases derive their specificity from sequence-specific protein-DNA contacts, but require more complex engineering strategies to generate protein structures that specifically bind to user-defined sequences area

Method used

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Embodiment

[0120] The invention is further described by the following examples, which do not limit the scope of the invention described in the claims.

[0121] Example #1: Sequence-specific nucleases regulated by epigenetics

[0122]A system was developed in which SpCas9 variants with R661A and Q695A mutations or with R661A or Q926A mutations were genetically fused to an engineered zinc finger array (ZF292R) targeting genomic integration of a single-copy EGFP reporter gene. Introduction of nuclease-induced DSBs into the EGFP coding region, followed by repair by NHEJ, can result in the introduction of frameshift mutations that result in cells becoming negative for EGFP, a phenotype that can be quantified using flow cytometry. We tested the nuclease activity of these variants with or without the ZF292R zinc finger array and with four different gRNA variants targeting the same site in EGFP: (1) with 20 nt homology to the target site and with Additional 5' gRNA with additional G (gRNA1) wit...

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Abstract

Methods and compositions for improving the specificity of genome-editing nucleases (e.g., RNA-guided CRISPR-Cas nucleases or engineered zinc finger nucleases) and customizable DNA-binding domain fusion proteins (e.g., RNA-guided dead-Cas9, RNA-guided dead-Cpf1, or engineered zinc finger arrays fused to transcriptional regulatory domains) for use as research reagents, in gene drives, or as therapeutic agents.

Description

[0001] priority statement [0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 408,645, filed October 14, 2016. The entire contents of the aforementioned documents are hereby incorporated by reference. [0003] Federally funded research or development [0004] This invention was made with US Government support under Grant Nos. DP1 GM105378 and R35GM118158 awarded by the National Institutes of Health. The US Government has certain rights in this invention. technical field [0005] Described herein are methods for improving genome editing nucleases (such as RNA-guided CRISPR-Cas nucleases or engineered zinc finger nucleases) and customizable DNA-binding domain fusion proteins (such as RNA-guided death cells fused to transcriptional regulatory domains). Cas9, RNA-guided dead Cpf1, or engineered zinc finger arrays) specific methods and compositions for use as research reagents, for gene drives, or as therapeutic agents. Background techniq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K38/54C12N15/87C12N15/00C07K14/00A61K39/00C07H21/04
CPCA61K38/54A61K48/00C12N9/22C12N15/102C12N15/87C07K2319/705C07K2319/715C12N15/90C07K2319/70C07K2319/81A61P31/00A61P43/00C12N2310/20C12N15/113C12N15/11C07K2319/80C12N15/63
Inventor J·K·乔昂格J·M·格尔克
Owner THE GENERAL HOSPITAL CORP
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