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Using split deaminases to limit unwanted off-target base editor deamination

A technology of deaminase and first division, applied in the direction of introducing foreign genetic material, enzymes, hydrolytic enzymes, etc. by using vectors

Pending Publication Date: 2020-05-01
THE GENERAL HOSPITAL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the efficiency of HDR may be substantially limited by non-homologous end joining-mediated repair of nuclease-induced breaks due to competition and more efficient induction of variable-length insertion / deletion mutations

Method used

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  • Using split deaminases to limit unwanted off-target base editor deamination
  • Using split deaminases to limit unwanted off-target base editor deamination
  • Using split deaminases to limit unwanted off-target base editor deamination

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Embodiment 1

[0228] Since a variety of mammalian deaminases can constitute functional BE proteins and multiple truncation points can generate functional split BEs, we sought to investigate a broad set of split BE candidate pairs (Table 2 above that could be adapted to List of representative deaminases for BE applications). Deaminase truncation points selected by evaluating structural information and identifying amino acid residues in the deaminase domain are unlikely to contribute to meaningful secondary structural components and thus are unlikely to affect the function of the intact enzyme . We selected six possible truncation regions for six mammalian deaminases (a full list of predicted partitions is included in Table 3). Each partition corresponds to a region of homology based on protein alignments in each deaminase listed. The sDA1 half-enzyme containing a truncated variant from partition X is called sDA1.X, and the sDA2 half-enzyme is similarly named.

[0229] Tables 4-6 show the ...

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Abstract

Described herein are methods and compositions for improving the genome-wide specificities of targeted base editing technologies. Herein, we describe dimeric base editing (BE) technologies that use split deaminases (sDA) that are functional when brought into close proximity to each other, one fused to a ZF and one to an nCas9-UGI protein comprising one or more UGIs, so as to limit the ability of the deaminase domain from deaminating at off-target ssDNA target sites independent of nCas9 R-loop formation. Thus, provided herein are fusion proteins comprising: (i) a first portion of a split deaminase ("sDAI") enzyme fused to a programmable DNA-binding domain; or (ii) a second portion of a split deaminase ("sDA2") fused to an nCas9 protein. The present invention also includes the vectors and cells comprising the vectors, as well as kits comprising the proteins and nucleic acids described herein.

Description

[0001] priority statement [0002] This application claims U.S. Provisional Patent Application Serial No. 62 / 511,296, filed May 25, 2017; U.S. Provisional Patent Application Serial No. 62 / 541,544, filed August 4, 2017; and U.S. Provisional Patent Application Serial No. 62 / 541,544, filed August 4, 2018 Benefit of Application Serial No. 62 / 622,676. The entire contents of the aforementioned documents are incorporated by reference. [0003] State-funded research or development [0004] This invention was made with US Government support under Grant No. GM118158 awarded by the National Institutes of Health. The US Government has certain rights in this invention. technical field [0005] Described herein are methods and compositions for improving the genome-wide specificity of targeted base editing technology. Background technique [0006] Base editing (BE) technology uses an engineered DNA-binding domain such as an RNA-guided, catalytically inactive Cas9 (dead Cas9 or dCas9), ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N9/22C12N15/10C12N15/63C12N15/85
CPCC12N9/22C12N9/78C12N15/10C12N15/102C12N15/63C12N15/90C12Y305/04001C07K2319/70C07K2319/80C12Q2521/539C12N2310/20C07K2319/81C07K14/435C07K14/4703C12N5/0647
Inventor J·K·乔昂格J·盎格斯特曼
Owner THE GENERAL HOSPITAL CORP
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