Tal-effector assembly platform, customized services, kits and assays
AssigneesGeneart AG, Life Technologies Corporation
InventorsFederico KATZEN, Jonathan Chesnut, Frank Notka, Matthias Arnold, Kevin Hoff, Stephen Whiteside
Life of this patent
Estimating the term…
Abstract
The invention generally relates to compositions and methods for designing and producing functional DNA binding effector molecules and associated customized services, tool kits and functional assays. In some aspects, the invention provides methods and tools for efficient assembly of customized TAL effector molecules. Furthermore, the invention relates to uses of TAL effector molecules and functional evaluation of such TAL by, for example, customized assays.
Claims28
A linear nucleic acid molecule comprising:
- (a) a region encoding an N-terminal portion of a TAL effector,
- (b) a region encoding a C-terminal portion of a TAL effector,
- (c) at least one recombination site, and
- (d) at least one covalently bound topoisomerase,
- wherein the topoisomerase is located at one of the termini of the linear nucleic acid molecule and is within 100 nucleotides of the at least one recombination site, and
- wherein, when the nucleic acid molecule is circularized and contains a TAL repeat located between the termini of the nucleic acid molecule, the circularized nucleic acid molecule encode a TAL effector which is capable binding to a specified nucleic acid sequence.
(canceled)
A method for preparing a TAL effector library, the method comprising:
- (a) connecting a population of TAL nucleic acid binding cassettes that individually encode adenine, guanine, thymidine, or cytosine base binders, when the base is present in a nucleic acid molecule, and
- (b) introducing the connected TAL nucleic acid binding cassettes generated in (a) into a vector to generate a TAL effector library,
- wherein the library encodes TAL effectors which bind to different nucleotide sequences.
(canceled)
A method for identifying TAL effectors that bind to specified nucleotide sequences, the method comprising:
- (a) connecting a population TAL nucleic acid binding cassettes which individually encode TAL subunits that bind to one of the bases adenine, guanine, thymidine, and cytosine, when the base is present in a nucleic acid molecule,
- (b) introducing the connected TAL nucleic acid binding cassettes generated in (a) into a vector to generate a TAL effector library, wherein the library contains TAL effectors which bind to different nucleotide sequences,
- (c) introducing the TAL effector library into a cell under conditions which allow for the expression of TAL effectors, and
- (d) screening the cells generated in (c) to identify cells in which at least one cellular parameter is altered by expression of a TAL effector.
A non-naturally occurring protein comprising:
- (a) an amine terminal region of between 25 and 500 amino acids,
- (b) a carboxyl terminal region of between 25 and 500 amino acids, and
- (c) a central region containing five or more amino acid segments which confer upon the non-naturally occurring protein sequence specific nucleic acid binding activity,
- wherein each of the individual amino acid segments in (c) are between 30 and 38 amino acid in length, and
- wherein at least one of the amino acid segments is at least 80% identical to one or more of the following amino acid sequences:
- (SEQ ID NO:
- 37)
- (1) FSQADIVKIAGN,
- (SEQ ID NO:
- 38)
- (2) GGAQALQAVLDLEP,
- (SEQ ID NO:
- 39)
- (3) GGAQALQAVLDLEPALRERG,
- (SEQ ID NO:
- 40)
- (4) FRTEDIVQMVS,
- (SEQ ID NO:
- 41)
- (5) GGSKNLAAVQA,
- (SEQ ID NO:
- 42)
- (6) GGSKNLEAVQA,
- (SEQ ID NO:
- 43)
- (7) LEPKDIVSIAS,
- (SEQ ID NO:
- 44)
- (8) GATQAITTLLNKW,
- (SEQ ID NO:
- 45)
- (9) GATQAITTLLNKWDXLRAKG,
- and
- (SEQ ID NO:
- 46)
- (10) GATQAITTLLNKWGXLRAKG;
- wherein X is one of the following amino acids:
- aspartic acid, serine, alanine, or glutamic acid.
Dependencies are inferred from the claim text.