# US 11,078,469 B2 · Evolution of TALENs > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,469 B2 - Document ID: US11078469B2 - Status: Granted - Kind: B2 - Application number: 16/804,228 - Filed: 2020-02-28 - Published: 2020-09-03 - Granted: 2021-08-03 - Earliest claimed: 2015-07-30 - Assignees: President and Fellows of Harvard College - Applicants: President and Fellows of Harvard College - Inventors: David R. Liu; Basil Hubbard; Ahmed Hussein Badran - Web page: https://patentgrep.com/patent/US11078469B2 ## CPC - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 15/62: DNA sequences coding for fusion proteins - C12N 15/8509: {for producing genetically modified animals, e.g. transgenic} - C12N 15/90: Stable introduction of foreign DNA into chromosome - A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid} - A61K 48/0091: {Purification or manufacturing processes for gene therapy compositions} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## 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. ## Claims ### Claim 1 A protein comprising a transcriptional activator-like effector (TALE) N-terminal domain having the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid sequence comprises an alanine to glutamic acid amino acid substitution at amino acid residue 39 of SEQ ID NO: 1, wherein the protein has DNA-binding activity. ### Claim 2 (depends on 1) The protein of claim 1 further comprising a lysine to glutamic acid substitution at amino acid residue 19 of SEQ ID NO: 1. ### Claim 3 (depends on 1) The protein of claim 1 further comprising a glycine to arginine amino acid substitution at amino acid residue 98 of SEQ ID NO: 1. ### Claim 4 A protein comprising a TALE N-terminal domain having the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid sequence comprises a lysine to glutamic acid substitution at amino acid residue 19 of SEQ ID NO: 1, wherein the protein has DNA-binding activity. ### Claim 5 (depends on 4) The protein of claim 4 further comprising a glycine to arginine amino acid substitution at amino acid residue 98 of SEQ ID NO: 1. ### Claim 6 (depends on 1) The protein of claim 1 further comprising one or more amino acid substitutions selected from the group consisting of S22N, G77D, A85T, T91A, A93G, P99S, P99T, A129E, and N136T of SEQ ID NO: 1. ### Claim 7 (depends on 1) The protein of claim 1 further comprising an arginine to tryptophan amino acid substitution at amino acid residue 21 of SEQ ID NO: 1. ### Claim 8 (depends on 4) The protein of claim 4 further comprising one or more amino acid substitutions selected from the group consisting of S22N, G77D, A85T, T91A, A93G, P99S, P99T, A129E, and N136T of SEQ ID NO: 1. ### Claim 9 (depends on 4) The protein of claim 4 further comprising an arginine to tryptophan amino acid substitution at amino acid residue 21 of SEQ ID NO: 1. ### Claim 10 (depends on 1) A method comprising contacting a nucleic acid molecule comprising a target sequence with the protein of claim 1 under conditions suitable for the protein to bind the target sequence. ### Claim 11 (depends on 10) The method of claim 10, wherein the contacting is in vitro. ### Claim 12 (depends on 10) The method of claim 10, wherein the contacting is in vivo. ### Claim 13 (depends on 10) The method of claim 10, wherein the nucleic acid molecule is in a cell. ### Claim 14 (depends on 13) The method of claim 13, wherein the cell is a mammalian cell. ### Claim 15 (depends on 4) A method comprising contacting a nucleic acid molecule comprising a target sequence with the protein of claim 4 under conditions suitable for the protein to bind the target sequence. ### Claim 16 (depends on 15) The method of claim 15, wherein the contacting is in vitro. ### Claim 17 (depends on 15) The method of claim 15, wherein the contacting is in vivo. ### Claim 18 (depends on 15) The method of claim 15, wherein the nucleic acid molecule is in a cell. ### Claim 19 (depends on 18) The method of claim 18, wherein the cell is a mammalian cell.