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Optimized endonucleases and uses thereof

一种内切核酸酶、多核苷酸的技术，应用在优化的内切核酸酶领域，能够解决非意图性脱靶作用、减少切割活性等问题

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

AI Technical Summary

Problems solved by technology

However, many meganucleases that have been specifically engineered for DNA cleavage have reduced cleavage activity relative to the naturally occurring meganuclease from which they were derived (US2010 / 0071083)
Most meganucleases also act on sequences similar to their optimal binding site, which may lead to unintended or even deleterious off-target effects

Method used

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  • Optimized endonucleases and uses thereof
  • Optimized endonucleases and uses thereof
  • Optimized endonucleases and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0326] Example 1: Construction of a sequence-specific DNA endonuclease expression cassette for expression in E. coli

Embodiment 1a

[0327] Example 1a: Basic construct

[0328] In this example, we show the general outline of the vector named "Construct I", which is suitable for transformation in E. coli. This general profile of the vector contains an ampicillin resistance gene for selection, an origin of replication for E. coli, and the gene araC (which encodes an arabinose-inducible transcriptional regulator). SEQ ID NO: 7 shows the stretch of "NNNNNNNNNN". This represents a placeholder for genes encoding different versions of sequence-specific DNA endonucleases. Different genes can be expressed from the arabinose-inducible pBAD promoter (Guzman et al., J Bacteriol 177:4121-4130 (1995)), and the gene sequences encoding different nuclease versions are given in the following examples.

[0329] The control construct in which the placeholder is replaced by the sequence of I-Scel (SEQ ID NO: 8) is called VC-SAH40-4.

Embodiment 2

[0330] Example 2: Escherichia coli plasmid encoding a stable version of nuclease

[0331] Different destabilizing sequences can be identified in the amino acid sequence of I-Scel. Among them are the weak C-terminal sequence of PEST containing amino acid residues 228 to 236, and the N-terminal sequence that shows similarity to the KEN motif (Pfleger and Kirschner, Genes and Dev. 14: 655-665 (2000)). According to the N-terminal rule, the second amino acid residue of I-Scel confers protein instability.

[0332] In order to test the influence of these sequences on the stability of nucleases, different versions of I-SceI were generated by PCR, which deleted the N-terminal amino acid, the C-terminal 9 amino acids, or both. These constructs are expressed according to "Construct I" described in Example 1a). Therefore, the placeholders are replaced by multiple sequences (shown in SEQ ID NO: 2, 3, 5) encoding each version of the nuclease. The plasmids are called VC-SAH43-8 (I-SceI with sh...

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Abstract

Provided are optimized endonucleases, as well as methods of targeted integration, targeted deletion or targeted mutation of [polynucleotides using optimized endonucleases.

Description

Invention field [0001] The present invention relates to an optimized endonuclease and a method for using the optimized endonuclease to perform targeted integration, targeted deletion or targeted mutation of polynucleotides. Background of the invention [0002] Genome engineering is a general term that summarizes different technologies for inserting, deleting, replacing or manipulating specific genetic sequences in the genome, which has a large number of therapeutic applications and biotechnological applications. All genome modification technologies more or less use recombinase, integrase or endonuclease to create DNA double-strand breaks at predetermined sites to promote homologous recombination. [0003] Although a large number of methods have been used to create DNA double-strand breaks, the development of effective methods to create DNA double-strand breaks at highly specific sites in the genome is still the main goal in gene therapy, agricultural technology, and synthetic biolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/11C12N15/63C07K19/00
CPCC12N9/22C12N5/14C12N15/82
Inventor A·赫鲁贝克C·比斯根
Owner BASF PLANT SCI GMBH
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