US 9,315,788 B2Granted

Method for the generation of compact TALE-nucleases and uses thereof

AssigneeCellectis

InventorsPhilippe Duchateau, Julien Valton, Claudia Bertonati, Jean-Charles Epinat, George H. Silva, Alexandre Juillerat, Marine Beurdeley

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Abstract

The present invention relates to a method for the generation of compact Transcription Activator-Like Effector Nucleases (TALENs) that can efficiently target and process double-stranded DNA. More specifically, the present invention concerns a method for the creation of TALENs that consist of a single TALE DNA binding domain fused to at least one catalytic domain such that the active entity is composed of a single polypeptide chain for simple and efficient vectorization and does not require dimerization to target a specific single double-stranded DNA target sequence of interest and process DNA nearby said DNA target sequence. The present invention also relates to compact TALENs, vectors, compositions and kits used to implement the method.

Claims33

Dependent claims

A compact Transcription Activator-Like Effector Nuclease (TALEN) monomer comprising:

(i) one core TALE scaffold comprising Repeat Variable Dipeptide regions (RVDs) having DNA binding specificity onto a specific double-stranded DNA target sequence of interest;
and
(ii) at least one I-TevI nuclease domain, wherein said I-TevI nuclease domain consists essentially of:
(a) amino acids 1-196 of SEQ ID NO:
20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-196 of SEQ ID NO:
20;
(b) amino acids 1-183 of SEQ ID NO:
20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-183 of SEQ ID NO:
20;
(c) amino acids 1-169 of SEQ ID NO:
20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-169 of SEQ ID NO:
20;
(d) amino acids 1-148 of SEQ ID NO:
20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-148 of SEQ ID NO:
20;
or
(e) amino acids 1-123 of SEQ ID NO:
20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-123 of SEQ ID NO:
20;
wherein said I-TevI nuclease domain is capable of cleaving DNA a few base pairs away from said double-stranded DNA target sequence of interest when fused to the C-terminus or N-terminus of said core TALE scaffold from (i);
wherein said compact TALEN monomer is assembled to bind said target DNA sequence and cleave double-stranded DNA without requiring dimerization.

23 more elements

A method for cleaving a double-stranded DNA, comprising:

(a) selecting one DNA target sequence of interest on one strand of a double-stranded DNA;
(b) providing a compact TALEN monomer comprising:
(i) one core TALE scaffold comprising Repeat Variable Dipeptide regions (RVDs) having DNA binding specificity onto the DNA target sequence of interest;
and
(ii) at least one I-TevI nuclease domain, wherein said I-TevI nuclease domain consists essentially of:
(a) amino acids 1-196 of SEQ ID NO:
20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-196 of SEQ ID NO:
20;
(b) amino acids 1-183 of SEQ ID NO:
20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-183 of SEQ ID NO:
20;
(c) amino acids 1-169 of SEQ ID NO:20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-169 of SEQ ID NO:
20;
(d) amino acids 1-148 of SEQ ID NO:
20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-148 of SEQ ID NO:
20;
or
(e) amino acids 1-123 of SEQ ID NO:
20;
or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino acid sequence identity with amino acids 1-123 of SEQ ID NO:
20;
wherein said I-TevI nuclease domain is capable of cleaving DNA a few base pairs away from said double-stranded DNA target sequence of interest when fused to the C-terminus or N-terminus of said core TALE scaffold from (i);
wherein said compact TALEN monomer is assembled to bind and cleave said double stranded DNA without requiring dimerization;
and
(c) contacting said double-stranded DNA with said compact TALEN monomer such that the double-stranded DNA is cleaved a few base pairs away in 3′ and/or 5′ direction(s) from said DNA target sequence of interest.

26 more elements

Dependencies are inferred from the claim text.

Facts

Application: 13/440,940Filed 5 Apr 2012 · us provisional application
Priority: 5 Apr 2011
Published: US 2013/0117869 A19 May 2013
Granted: 19 Apr 2016
Term ends: Not estimatedThe source records don’t support an estimate.

Classification18 codes

C12N 9/22
Acting on ester bonds: ribonucleases [RNase]; Deoxyribonucleases [DNase]
A61P 43/00
Drugs for specific purposes, not provided for in groups a61p1/00-a61p41/00
C07K 14/195
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: from bacteria
C12N 5/0602
Animal cells or tissues; Human cells or tissues: vertebrate cells

Family

Application61/472,065Us provisional application · filed 5 Apr 2011
Application61/496,454Us provisional application · filed 13 Jun 2011
Application61/499,043Us provisional application · filed 20 Jun 2011
Application61/499,047Us provisional application · filed 20 Jun 2011
Application61/533,098Us provisional application · filed 9 Sep 2011
Application61/533,123Us provisional application · filed 9 Sep 2011
Application61/579,544Us provisional application · filed 22 Dec 2011
US 2013/0117869 A1Pre-grant publication · published 9 May 2013
US 9,315,788 B2This patent

From the US filing history. Foreign family members aren’t listed.