Transcription activator-like effectors

Patent metadata

Field	Value
Publication number	US-9499592-B2
Application number	US-201213353662-A
Country	US
Kind code	B2
Filing date	Jan 19, 2012
Priority date	Jan 26, 2011
Publication date	Nov 22, 2016
Grant date	Nov 22, 2016

Abstract

Provided herein are compositions, kits and methods useful in the construction of designer transcription activator-like effector (dTALE) polypeptides.

First claim

We claim:

1. A nucleic acid molecule encoding a designer transcription activator-like effector (dTALE) polypeptide fragment, the nucleic acid molecule comprising a sequence encoding a nucleic acid binding domain of the dTALE polypeptide fragment and one or more mammalian effector domains,
wherein the sequence encoding the nucleic acid binding domain comprises a sequence encoding two or more monomer units arranged in a predetermined 5′ to 3′ non-endogenous TALE order,
wherein each said monomer unit comprises a variable diresidue that is capable of specifically binding a target nucleotide,
wherein the nucleic acid binding domain encoded by the nucleic acid molecule is capable of specifically binding a predetermined target nucleic acid sequence,
wherein each of the one or more mammalian effector domains encoded by the nucleic acid molecule is capable of mediating an effector function, and
wherein the nucleic acid molecule further comprises an expression vector comprising the sequence of an expression vector of SEQ ID NOs: 192-195.

2. The nucleic acid molecule of claim 1 , wherein the two or more monomer units of the sequence comprise:
a) the monomer units of a TALE polypeptide of SEQ ID NOs: 4-167; the monomer units encoded by the nucleic acid sequences of SEQ ID NOs: 168-171; or the monomer units of SEQ ID NOs: 172-191; or
b) an amino acid sequence that is at least 70% identical to: the monomer units of a TALE polypeptide of SEQ ID NOs: 4-167; the monomer units encoded by the nucleic acid sequences of SEQ ID NOs: 168-171; or the monomer units of SEQ ID NOs: 172-191.

3. The nucleic acid molecule of claim 1 , wherein the predetermined target nucleic acid sequence to which the nucleic acid binding domain specifically binds comprises a bacterial, protozoan, fungal, animal, or viral nucleic acid sequence.

4. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule further comprises at least one nucleic acid sequence:
a) of an expression vector;
b) of a nuclear localization signal;
c) encoding an N-terminal domain that is at least 70% identical to an amino acid sequence of an N-terminal domain from a transcription activator-like effector (TALE) polypeptide from a bacterium of the genus Xanthomonas , or a fragment thereof, and wherein the at least one nucleic acid sequence encoding the N-terminal domain is 5′ of the sequence encoding the nucleic acid binding domain of the dTALE polypeptide;
d) encoding a C-terminal domain that is at least 70% identical to an amino acid sequence of a C-terminal domain from a transcription activator-like effector (TALE) polypeptide from a bacterium of the genus Xanthomonas , or a fragment thereof, and wherein the at least one nucleic acid sequence encoding the C-terminal domain is 3′ of the sequence encoding the nucleic acid binding domain of the dTALE polypeptide; or
e) any combination thereof.

5. The nucleic acid molecule of claim 4 , wherein the nucleic acid molecule comprises: a sequence encoding an N-terminal domain that is at least 70% identical to the amino acid sequence of an N-terminal domain sequence from a transcription activator-like effector (TALE) polypeptide from a bacterium of the genus Xanthomonas , or a fragment thereof, and wherein the sequence encoding the N-terminal domain is 5′ of the sequence encoding the nucleic acid binding domain of the dTALE polypeptide; a sequence encoding a C-terminal domain that is at least 70% identical to the amino acid sequence of a C-terminal domain from a transcription activator-like effector (TALE) polypeptide from a bacterium of the genus Xanthomonas , or a fragment thereof, and wherein the sequence encoding the C-terminal domain is 3′ of the sequence encoding the nucleic acid binding domain of the dTALE polypeptide; or a combination thereof, and the TALE polypeptide from a bacterium of the genus Xanthomonas comprises a sequence of SEQ ID NOs: 4-167.

6. The nucleic acid molecule of claim 1 , wherein the variable diresidue of at least one of the monomer units is an engineered variable diresidue capable of specifically binding a predetermined target nucleotide.

7. The nucleic acid molecule of claim 1 , wherein the sequence encoding two or more monomer units is an engineered sequence capable of minimizing sequence repetitiveness among the monomer units encoded by the nucleic acid molecule.

8. The nucleic acid molecule of claim 1 , wherein a monomer unit encoded at the 5′ end of the nucleic acid molecule specifically binds to a thymine nucleotide.

9. The nucleic acid molecule of claim 6 , wherein the variable diresidue of at least one of the monomer units encoded by the nucleic acid molecule is an engineered variable diresidue capable of specifically binding the predetermined target nucleotide by encoding NG for specifically binding thymine, HD for specifically binding cytosine, NI for specifically binding adenine, or NN for specifically binding guanine.

10. The nucleic acid molecule of claim 1 , wherein the sequence encoding the two or more monomer units is contiguous and does not comprise insertion or deletion of nucleic acid sequences.

11. The nucleic acid molecule of claim 4 , wherein the expression vector, the one or more mammalian effector domains, the nuclear localization signal, or a combination thereof has activity in a host cell that is not a plant cell.

12. The nucleic acid molecule of claim 1 , wherein the effector function comprises a nuclease function, recombinase function, epigenetic modifying function, transposase function, integrase function, resolvase function, invertase function, protease function, DNA methyltransferase function, DNA demethylase function, histone acetylase function, histone deacetylase function, transcriptional repressor function, transcriptional activator function, DNA binding protein function, transcription factor recruiting protein function, nuclearlocalization signal function, cellular uptake signal activity function, or any combination thereof.

Assignees

• Zhang Feng
• Cong Le
• Kosuri Sriram
• Church George M
• Harvard College

Inventors

• Zhang Feng
• Cong Le
• Kosuri Sriram
• Church George M

Classifications

• C07K14/195 Primary

  from bacteria · CPC title

• C07K14/4705

  stimulating, promoting or activating activity · CPC title

• C07K2319/09

  containing a nuclear localisation signal · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title