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Recognition sequences for i-crei-derived meganucleases and uses thereof

a meganuclease and recognition sequence technology, applied in the field of molecular biology and recombinant nucleic acid technology, can solve the problems of residual non-specific cleavage activity, high mutagenic and toxic, and inability to target gene modifications to unique sites within a chromosomal background, and achieve the effect of more efficient cleavag

Inactive Publication Date: 2011-08-18
PRECISION BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]In one aspect, the invention provides methods of identifying sets of 22 base pair DNA sequences which can be cleaved by I-CreI-derived meganucleases and which have, at their center, one of a limited set of four base pair DNA center sequences that contribute to more efficient cleava...

Problems solved by technology

Although these methods efficiently stimulate recombination, the double-stranded breaks are randomly dispersed in the genome, which can be highly mutagenic and toxic.
At present, the inability to target gene modifications to unique sites within a chromosomal background is a major impediment to successful genome engineering.
Although these artificial zinc finger nucleases stimulate site-specific recombination, they retain residual non-specific cleavage activity resulting from under-regulation of the nuclease domain and frequently cleave at unintended sites (Smith et al.
Such unintended cleavage can cause mutations and toxicity in the treated organism (Porteus et al.
Natural meganucleases, primarily from the LAGLIDADG family, have been used to effectively promote site-specific genome modification in plants, yeast, Drosophila, mammalian cells and mice, but this approach has been limited to the modification of either homologous genes that conserve the meganuclease recognition sequence (Monnat et al.

Method used

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  • Recognition sequences for i-crei-derived meganucleases and uses thereof
  • Recognition sequences for i-crei-derived meganucleases and uses thereof
  • Recognition sequences for i-crei-derived meganucleases and uses thereof

Examples

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Effect test

example 1

Cleavage of an Optimized Meganuclease Recognition Site by a Rationally-Designed, I-CreI-Derived Meganuclease Homodimer in vivo

[0106]An engineered meganuclease called BRP2 (SEQ ID NO: 8) was produced using the method disclosed in WO 2007 / 047859. This meganuclease is derived from I-CreI and was engineered to recognize DNA sites that are not recognized by wild-type I-CreI (e.g., BRP2 recognition sequences include SEQ ID NO: 9 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 12). To facilitate nuclear localization of the engineered meganuclease, an SV40 nuclear localization signal (NLS, SEQ ID NO: 13) was added to the N-terminus of the protein. Conventional Agrobacterium-mediated transformation procedures were used to transform Arabidopsis thaliana with a T-DNA containing a codon-optimized BRP2 coding sequence (SEQ ID NO: 14). Expression of BRP2 meganuclease was under the control of a Hsp70 promoter and a NOS terminator. A pair of BRP2 recognition sequences were housed on the same T-D...

example 2

Cleavage of an Optimized Meganuclease Recognition Site by a Rationally-Designed, I-CreI-Derived Meganuclease Single-Chain Heterodimer in vivo

[0108]The engineered meganuclease BRP12-SC (SEQ ID NO: 15) was produced in accordance with WO 2007 / 047859, except that this meganuclease is a single-chain heterodimer. As discussed in WO 2007 / 047859, wild-type I-CreI binds to and cleaves DNA as a homodimer. As a consequence, the natural recognition sequence for I-CreI is pseudo-palindromic. The BRP12-SC recognition sequences, however, are non-palindromic (e.g., SEQ ID NO: 16 and SEQ ID NO: 17, or SEQ ID NO: 18 and SEQ ID NO: 19). This necessitates the use of an engineered meganuclease heterodimer comprising a pair of subunits each of which recognizes one half-site within the full-length recognition sequence. In the case of BRP12-SC, the two engineered meganuclease monomers are physically linked to one another using an amino acid linker to produce a single-chain heterodimer. This linker comprise...

example 3

Cleavage of an Optimized Meganuclease Recognition Site by a Rationally-Designed, I-CreI-Derived Meganuclease Homodimer In Vitro

[0111]The BRP2 meganuclease described in Example 1 (SEQ ID NO: 8) was expressed in E. coli and purified as in Example 1 of WO 2007 / 047859. The purified meganuclease was then added at varying concentrations to reactions containing plasmids harboring BRP2 recognition sequences with either a GTAC or TAGA center sequence (0.25 picomoles of plasmid substrate in 25 microliters of SA buffer: 25 mM Tris-HCL, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 5 mM EDTA). Reactions were incubated at 37° C. for 2 hours and were then visualized by gel electrophoresis and the percentage of each plasmid substrate cleaved by the meganuclease was plotted as a function of meganuclease concentration (FIG. 5). It was found that the plasmid substrate with the TAGA center sequence was cleaved by the meganuclease in vitro, but that cleavage of this substrate required a far higher concentration of ...

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Abstract

Methods of cleaving double-stranded DNA that can be recognized and cleaved by a rationally-designed, I-CreI-derived meganuclease are provided. Also provided are recombinant nucleic acids, cells, and organisms containing such recombinant nucleic acids, as well as cells and organisms produced using such meganucleases. Also provided are methods of conducting a custom-designed, I-CreI-derived meganuclease business.

Description

REFERENCE TO RELATED APPLICATIONS[0001]This application is a Continuation of International Application PCT / US2009 / 50566 filed on Jul. 14, 2009, which claims the benefit of U.S. Provisional Application No. 61 / 080,453, filed Jul. 14, 2008, the entire disclosures of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention relates to the field of molecular biology and recombinant nucleic acid technology. In particular, the invention relates to DNA sequences that can be recognized and cleaved by a non-naturally-occurring, rationally-designed, I-CreI-derived homing endonuclease and methods of using same. The invention also relates to methods of producing recombinant nucleic acids, cells, and organisms using such meganucleases which cleave such DNA sites. The invention further relates to methods of conducting a custom-designed, I-CreI-derived meganuclease business.BACKGROUND OF THE INVENTION[0003]Genome engineering requires the ability to insert, delete, substit...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12N5/10C12N1/15C12N1/21G06Q90/00
CPCC12N9/22C12Q1/6811G06Q99/00
Inventor JANTZ, DEREKSMITH, JAMES J.
Owner PRECISION BIOSCI
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