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Chimeric endonucleases and uses thereof

A technology of endonuclease and polynucleotide, applied in the field of chimeric endonuclease, can solve problems such as limiting off-target effects

Inactive Publication Date: 2012-10-31
BASF PLANT SCI GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a need in the art to develop meganucleases with high induction efficiency for homologous recombination and / or high specificity for their binding sites, thereby limiting the risk of off-target effects

Method used

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  • Chimeric endonucleases and uses thereof
  • Chimeric endonucleases and uses thereof
  • Chimeric endonucleases and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0365] Preparation of Chimeric Endonucleases

[0366] Endonucleases and heterologous DNA binding domains can be combined in a number of alternative ways.

[0367] For example, more than one endonuclease can be combined with one or more heterologous DNA binding domains, or more than one heterologous DNA binding domain can be combined with one endonuclease. It is also possible to combine more than one endonuclease with more than one heterologous DNA binding domain.

[0368] The heterologous DNA binding domain can be fused to the N-terminal end or the C-terminal end of the endonuclease. One or more heterologous DNA binding domains can also be fused to the N-terminal end of the endonuclease and one or more heterologous DNA binding domains can be fused to the C-terminal end of the endonuclease. Alternating combinations of endonucleases and heterologous DNA binding domains can also be made.

[0369] Where chimeric endonucleases contain more than one endonuclease or more than on...

Embodiment 1

[0604] Example 1: Constructs with sequence-specific DNA endonuclease expression cassettes for expression in E. coli

Embodiment 1a

[0605] Example 1a: Basic constructs

[0606] In this example, we show a general outline of a vector designated "Construct I", which is suitable for transformation in E. coli. This general outline of the vector contains the ampicillin resistance gene for selection, the origin of replication for E. coli and the gene araC (which encodes an arabinose-inducible transcriptional regulator). Different genes encoding different versions of sequence-specific DNA endonucleases can be expressed from the arabinose-inducible pBAD promoter (Guzman et al., J Bacteriol 177:4121-4130 (1995)). Gene sequences encoding different nuclease versions are given in the Examples below.

[0607] The control construct in which the sequence encoding I-SceI (SEQ ID NO: 22) was designated VC-SAH40-4.

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Abstract

The invention relates to chimeric endonucleases, comprising an endonuclease and a heterologous DNA binding domain, as well as methods of targeted integration, targeted deletion or targeted mutation of polynucleotides using chimeric endonucleases.

Description

field of invention [0001] The present invention relates to chimeric endonucleases comprising an endonuclease and a heterologous DNA binding domain, and methods of using chimeric endonucleases for targeted integration, targeted deletion or targeted mutation of polynucleotides. Background of the invention [0002] Genome engineering is a general term summarizing different techniques for inserting, deleting, substituting or manipulating specific genetic sequences within the genome, which has numerous therapeutic and biotechnological applications. All genome engineering techniques use more or less recombinases, integrases or endonucleases to create DNA double-strand breaks at predetermined sites to facilitate homologous recombination. [0003] Although a large number of methods have been utilized to create DNA DSBs, developing efficient methods to create DNA DSBs at highly specific sites in the genome remains a major goal in gene therapy, agricultural technology, and synthetic b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/10C12N15/52C12N15/62C12N15/82
CPCC12N15/1082C07K2319/81C12N15/8213C07K2319/80C12N9/22A61P43/00
Inventor A·赫鲁贝克C·比斯根H·W·霍夫肯
Owner BASF PLANT SCI GMBH
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