US 2024/0200044 A1 · EVOLUTION OF TALENS

Abstract

Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5′ nucleotide of the target is specific for thymine (T). TALE domains with alternative 5′ nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. Another drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities. Some aspects of this disclosure provide engineered TALE domains and TALEs comprising such engineered domains, e.g., TALE nucleases (TALENs), TALE transcriptional activators, TALE transcriptional repressors, and TALE epigenetic modification enzymes, with altered 5′ nucleotide specificities of target sequences. Engineered TALEs that target ATM with greater specificity are also provided.

Claims130

Dependent claims

A protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO:

1, wherein the amino acid sequence comprises an alanine to glutamic acid amino acid substitution at amino acid residue 39 (A39E) of SEQ ID NO:
1 or a homologous residue in a canonical N-terminal TALE domain, and/or a lysine to glutamic acid substitution at amino acid residue 19 (K19E) of SEQ ID NO:1 or a homologous residue in a canonical N-terminal TALE domain.

A method of phage-assisted, continuous evolution of a DNA binding domain, the method comprising

(a) contacting a flow of host cells through a lagoon with a selection phage comprising a nucleic acid sequence encoding a DNA-binding domain to be evolved, and
(b) incubating the selection phage or phagemid in the flow of host cells under conditions suitable for the selection phage to replicate and propagate within the flow of host cells, and for the nucleic acid sequence encoding the DNA-binding domain to be evolved to mutate;
wherein the host cells are introduced through the lagoon at a flow rate that is faster than the replication rate of the host cells and slower than the replication rate of the phage, thereby permitting replication and propagation of the selection phage in the lagoon;
and
wherein the flow of host cells comprises a plurality of host cells harboring a positive selection construct comprising a nucleic acid sequence encoding a gene product essential for the generation of infectious phage particles, wherein the gene product essential for the generation of infectious phage particles is expressed in response to a desired DNA-binding activity of the DNA-binding domain to be evolved or an evolution product thereof;
wherein the selection phage does not comprise a nucleic acid sequence encoding the gene product essential for the generation of infectious phage particles;
and
wherein the flow of host cells comprises a plurality of host cells harboring a negative selection construct comprising a nucleic acid sequence encoding a dominant negative gene product that decreases or abolishes the production of infectious phage particles, wherein the dominant negative gene product is expressed in response to an undesired activity of the DNA-binding domain to be evolved or an evolution product thereof.

5 more elements

Dependencies are inferred from the claim text.

Evolution of talens

Life of this patent

Evolution of talens

Life of this patent

Abstract

Claims130