# US 2024/0200044 A1 · Evolution of talens

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- Number: US 2024/0200044 A1
- Document ID: US20240200044A1
- Status: Published application
- Kind: A1
- Application number: 18/414,114
- Filed: 2024-01-16
- Published: 2024-06-20
- 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/US20240200044A1

## 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 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.

### Claim 2 (depends on 1)

The protein of claim 1, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the amino acid sequence provided in SEQ ID NO: 1.

### Claim 3 (depends on 1, 2)

The protein of claim 1 or 2, wherein the amino acid sequence comprises an alanine to glutamic acid substitution at amino acid residue 93 (A93E) of SEQ ID NO: 1 or a homologous residue in a canonical N-terminal TALE domain.

### Claim 4 (depends on 1, 2, 3)

The protein of any one of claims 1-3, wherein the amino acid sequence comprises a glycine to arginine amino acid substitution at amino acid residue 98 (G98R) of SEQ ID NO: 1 or a homologous residue in a canonical N-terminal TALE domain.

### Claim 5 (depends on 1, 2, 3, 4)

The protein of any one of claims 1-4, wherein the amino acid sequence comprises 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 or a homologous residue in a canonical N-terminal TALE domain.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The protein of any one of claims 1-5, wherein the amino acid sequence comprises an arginine to tryptophan amino acid substitution at amino acid residue 21 (R21W) of SEQ ID NO: 1 or a homologous residue in a canonical N-terminal TALE domain.

### Claim 7

A protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence LTPX1QVVAIAX2X3X4GGX5X6ALETVQRLLPVLCQX7HG (SEQ ID NO: 2), wherein
X1 is D, E or A, wherein X2 is S or N, wherein X3 is N or H, wherein X4 is G, D, 1, or N, wherein X5 is K or R, wherein X6 is Q or P, wherein X7 is D or A, and
wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of T2A, P3L, P3S, X14G, X14K, X14N, X211K, X211Y, X312H, X413K, X413H, G15S, X516R, X617P, T21A, L26F, P27S, V28G, Q31K, X732S, D32E, and H33L.

### Claim 8 (depends on 7)

The protein of claim 7, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

### Claim 9 (depends on 7, 8)

The protein of claim 7 or 8, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of P3L, X14G, X14K, X211Y, X516R, X617P, T21A, and L26F.

### Claim 10 (depends on 7, 8, 9)

The protein of any of claims 7-9, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of P3S, X14K, X312H, X516R, and L26F.

### Claim 11 (depends on 7, 8, 9, 10)

The protein of any of claims 7-10, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of X14N, X14K, X211K, G15S, X516R, L26F, P27S, A32S, D32E, and H33L.

### Claim 12 (depends on 7, 8, 9, 10, 11)

The protein of any of claims 7-11, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of T2A, P3L, X14K, X312H, V28G, and Q31K.

### Claim 13 (depends on 7, 8, 9, 10, 11, 12)

The protein of any of claims 7-12, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of X14N, X14K, X211K, X516R, T21A, and L26F.

### Claim 14 (depends on 7, 8, 9, 10, 11, 12)

The protein of any of claims 7-12, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of A8G, X413K, X413H, A18G, E20G, Q23K, L26A, H33P, H33Y, and 034S.

### Claim 15 (depends on 7, 8, 9, 10, 11, 12, 13, 14)

The protein of any of claims 7-14, wherein the protein comprises a plurality of amino acid sequences that are at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

### Claim 16 (depends on 15)

The protein of claim 15, wherein the plurality of amino acid sequences comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 amino acid sequences.

### Claim 17 (depends on 15, 16)

The protein of claim 15 or 16, wherein the plurality of amino acid sequences are directly adjoined to each other without a linker.

### Claim 18 (depends on 15, 16, 17)

The protein of any of claims 15-17, wherein the plurality of amino acid sequences form a TALE repeat array.

### Claim 19 (depends on 15)

The protein of claim 15, wherein the plurality of amino acid sequences are from a TALE repeat array.

### Claim 20 (depends on 15, 16, 17, 18, 19)

The protein of any of claims 15-19, wherein the amino acid sequence is at least 80% identical to the amino acid sequence of SEQ ID NO: 6, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of K16R, K50R, L94F, T104A, P173L, L196F, K220R, L230F, A236S, N249Y, Q255P, T259A, D276G, L332F, Q337K, H373L, P377L, N386H, G389S, P401S, D406E, P41 IS, D412N, V436G, E446K, N453K, N455K, K458R, and P513L.

### Claim 21 (depends on 20)

The protein of claim 20, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the amino acid sequence provided in SEQ ID NO: 6.

### Claim 22 (depends on 20, 21)

The protein of claim 20 or 21, wherein the amino acid sequence comprises amino acid substitutions
(a) K50R and L230F;
(b) L230F;
(c) L230F and N249Y;
(d) Q255P;
(e) T259A;
(f) D276G, E446K, and P513L; or
(g) P377L.

### Claim 23 (depends on 20, 21)

The protein of claim 20 or 21, wherein the amino acid sequence comprises amino acid substitutions
(a) K50R and N453K;
(b) L332F and K458R;
(c) N386H;
(d) P411S and N453K;
(e) N453K;
(f) E446K; or
(g) K458R.

### Claim 24 (depends on 20, 21)

The protein of claim 20 or 21, wherein the amino acid sequence comprises amino acid substitutions
(a) K16R, G389S, and E446K;
(b) L94F;
(c) L196F, G389S, P401S, and E446K;
(d) K220R;
(e) A236S, G389S, and E446K;
(f) H373L and D412N;
(g) G389S, D406E, and E446K;
(h) D412N; or
(i) N455K.

### Claim 25 (depends on 20, 21)

The protein of claim 20 or 21, wherein the amino acid sequence comprises amino acid substitutions
(a) T104A, Q337K, N386H, and E446K;
(b) P173L, Q337K, N386H, E446K, and V436G; or
(c) Q337K, N386H, E446K, and V436G.

### Claim 26

A protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 3, 4, or 5, wherein the amino acid sequence comprises a glutamine to proline amino acid substitution at amino acid residue 5 (Q5P) as compared to either SEQ ID NO: 3, 4, or 5, or a homologous residue in a canonical C-terminal TALE domain.

### Claim 27 (depends on 26)

The protein of claim 26, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the amino acid sequence provided in SEQ ID NO: 3, 4, or 5.

### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

A protein comprising the structure
[N-terminal domain]-[TALE repeat array]-[C-terminal domain];
wherein the N-terminal domain comprises the protein of any of claims 1-6;
the TALE repeat array comprises the protein of any of claims 7-25;
and/or the C-terminal domain comprises the protein of any of claims 26-27.

### Claim 29 (depends on 28)

The protein of claim 28 further comprising an effector domain.

### Claim 30 (depends on 28, 29)

The protein of claim 28 or 29 comprising the structure [N-terminal domain]-[TALE repeat array]-[C-terminal domain]-[effector domain], or [effector domain]-[N-terminal domain]-[TALE repeat array]-[C-terminal domain].

### Claim 31 (depends on 28, 29, 30)

The protein of any of claims 28-30, wherein the effector domain comprises a nuclease domain, a transcriptional activator or repressor domain, a recombinase domain, or an epigenetic modification enzyme domain.

### Claim 32 (depends on 28, 29, 30, 31)

The protein of any of claims 28-31, wherein the effector domain comprises a FokI nuclease domain.

### Claim 33 (depends on 28, 29, 30, 31, 32)

The protein of any of claims 28-32, wherein the effector domain is an RNA polymerase domain.

### Claim 34 (depends on 33)

The protein of claim 33, wherein the RNA polymerase domain is an RNAPω domain.

### Claim 35 (depends on 33, 34)

The protein of claim 33 or 34, wherein the RNA polymerase domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 10.

### Claim 36 (depends on 34, 35)

The protein of claim 34 or 35, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 100% identical to the amino acid sequence provided in SEQ ID NO: 10.

### Claim 37 (depends on 35, 36)

The protein of claim 35 or 36, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of V9G, D17G, A26V, M29T, P36S, E52G, E52-, and V38G.

### Claim 38 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

The protein of any of claims 28-37, further comprising a linker, an epitope tag and/or a nuclear localization sequence (NLS).

### Claim 39 (depends on 38)

The protein of claim 38, wherein the linker is an amino acid linker.

### Claim 40 (depends on 39)

The protein of claim 39, wherein the amino acid linker comprises at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, or 100 amino acids.

### Claim 41 (depends on 38, 39, 40)

The protein of any of claims 38-40, wherein the linker comprises the amino acid sequence provided in SEQ ID NO: 8.

### Claim 42 (depends on 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)

The protein of any of claims 29-41, wherein the linker is positioned between the C-terminal domain and the effector domain.

### Claim 43 (depends on 38)

The protein of claim 38, wherein the epitope tag is a FLAG tag.

### Claim 44 (depends on 38, 39, 40, 41, 42, 43)

The protein of any of claims 38-43, wherein the epitope tag and/or the NLS is positioned at the N-terminus of the N-terminal domain.

### Claim 45 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)

The protein of any of claims 28-44, further comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 9.

### Claim 46 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)

The protein of any of claims 28-45, further comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 100% identical to the amino acid sequence provided in SEQ ID NO: 9.

### Claim 47 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)

The protein of any of claims 28-46 comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 11, wherein the amino acid sequence comprises one or more of amino acid substitutions
(a) A79E and Q711P;
(b) A79E and L406F;
(c) A79E, L406F, and N425Y;
(d) A79E, P553L, and Q711P;
(e) A79E, K226R, L406F, and Q711P;
(f) Q431P and Q711P;
(h) Q431P, Q711P, and P765S;
(i) T435A and Q711P; or
(j) D452G, E622K, and P689L.

### Claim 48 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)

The protein of any of claims 28-46 comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 11, wherein the amino acid sequence comprises one or more of amino acid substitutions
(a) A79E and N562H;
(b) A79E, L508F, and K634R;
(c) G138R and N629K;
(d) K226R and N629K;
(e) L508F and K634R;
(f) P587S and N629K;
(g) E622K; or
(h) K634R.

### Claim 49 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)

The protein of any of claims 28-46 comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 11, wherein the amino acid sequence comprises one or more of amino acid substitutions
(a) S62N, A125T, A412S, G565S, E622K, and V738G;
(b) Gi 17D, A169E, G565S, D582E, E622K, and V738G;
(c) T131A, P139S, N176T, K192R, G565S, E622K, and V738G;
(d) T131A, P139T, N176T, K192R, G565S, E622K, and V738G;
(e) A133E, K396R, and N683K;
(f) A133G, K396R, and N683K;
(g) A133E, H549L, D588N, and V738G;
(h) A133G, H549L, D588N, and V738G;
(i) A133E, D588N, and V738G;
(j) A133G, D588N, and V738G;
(k) P139S, L372F, G565S, P577S, E622K, and V7380;
(l) P139T, L372F, G565S, P577S, E622K, and V738G;
(m) K192R, G565S, E622K, and V738G;
(n) L270F, N629K, and V738G; or
(o) N631K and V738G.

### Claim 50 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)

The protein of any of claims 28-46 comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 11, wherein the amino acid sequence comprises one or more of amino acid substitutions
(a) K27N, K59E, Q513K, N562H, E622K, V612G, Q711P, M758T, and V767G;
(b) K27N, K59E, P349L, Q513K, N562H, E622K, V612G, Q711P, M758T, and V767G;
(c) K59E, T280A, Q513K, N562H, E622K, Q711P, D746G, and V767G; or
(d) K59E, R61W, T280A, Q513K, N562H, E622K, Q711P, D746G, and V767G.

### Claim 51 (depends on 47, 48, 49, 50)

The protein of any of claims 47-50, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 100% identical to the amino acid sequence provided in SEQ ID NO: 11.

### Claim 52

A protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 12, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of A76T, K84R, D134E, L162S, A222S, K288R, Q329K, R330K, A338T, A392V, A416V, P435Q, V464I, L468F, and K512R.

### Claim 53 (depends on 52)

The protein of claim 52, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 100% identical to the amino acid sequence provided in SEQ ID NO: 12.

### Claim 54 (depends on 52, 53)

The protein of claim 52 or 53, wherein the amino acid sequence comprises amino acid substitutions
(a) A76T;
(b) A76T and Q329K;
(c) A76T, L162S, and Q329K; or
(d) K84R, A222S, A338T, and A416V.

### Claim 55 (depends on 52, 53)

The protein of claim 52 or 53, wherein the amino acid sequence comprises amino acid substitutions
(a) A76T;
(b) A76T, K288R, and A392V;
(c) D134E, V464I, and L468F;
(d) A222S, A338T, A416V, and P435Q; or
(e) A222S, A338T, A416V, and K512R.

### Claim 56 (depends on 52, 53)

The protein of claim 52 or 53, wherein the amino acid sequence comprises amino acid substitutions
(a) A76T, Q329K, and R330K;
(b) A76T and Q329K;
(c) A76T, L162S, and Q329K; or
(d) A76T and Q329K.

### Claim 57 (depends on 52, 53)

The protein of claim 52 or 53 wherein the amino acid sequence comprises amino acid substitutions
(a) A76T; or
(b) A76T, L162S, and Q329K.

### Claim 58

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 one or more amino acid substitutions selected from the group consisting of Q13R, A25E, W126C, and G132R, or a homologous residue in a canonical N-terminal TALE domain.

### Claim 59 (depends on 58)

The protein of claim 58, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 100% identical to the amino acid sequence provided in SEQ ID NO: 1.

### Claim 60 (depends on 52, 53, 54, 55, 56, 57, 58, 59)

The protein of any of claims 52-59 comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 13, wherein the amino acid sequence comprises amino acid substitutions
(a) Q53R and A252T;
(b) W166C, K260R, A398S, A514T, A592V, and Q745P;
(c) A252T, Q505K, and Q745P; or
(d) A252T, L338S, Q505K, and Q745P.

### Claim 61 (depends on 52, 53, 54, 55, 56, 57, 58, 59)

The protein of claims 52-59 comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 13, wherein the amino acid sequence comprises amino acid substitutions
(a) A65E and E815G;
(b) W166C, A398S, A514T, A592V, and K688R;
(c) W166C, A398S, A514T, A592V, and P611Q;
(d) G172R and A252T;
(e) A252T, K464R, and A568V; or
(f) D310E, V6401, and L644F.

### Claim 62 (depends on 52, 53, 54, 55, 56, 57, 58, 59)

The protein of claims 52-59 comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 13, wherein the amino acid sequence comprises amino acid substitutions
(a) A252T; Q505K; R506K; Q745P, and A789V;
(b) A252T; Q505K; Q745P, and A789V; or
(c) A252T; L338S; Q505K; Q745P, and A789V.

### Claim 63 (depends on 52, 53, 54, 55, 56, 57, 58, 59)

The protein of claims 52-59 comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 13, wherein the amino acid sequence comprises amino acid substitutions
(a) Q53R and A252T; or
(b) A252T, L338S, Q505K, and Q745P.

### Claim 64 (depends on 60, 61, 62, 63)

The protein of any of claims 60-63, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 100% identical to the amino acid sequence provided in SEQ ID NO: 13.

### Claim 65 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)

The protein of any of claims 1-64, wherein the protein comprises a TALE repeat array that binds a target sequence comprised in a genome.

### Claim 66 (depends on 65)

The protein of claim 65, wherein the target sequence is comprised in or is in proximity to a gene known to be associated with a disease or disorder.

### Claim 67 (depends on 65)

The protein of claim 65, wherein the target sequence is comprised in or is in proximity to a regulatory sequence of a gene known to be associated with a disease or disorder.

### Claim 68 (depends on 66, 67)

The protein of claim 66 or 67, wherein the disease or disorder is a proliferative disease or disorder.

### Claim 69 (depends on 68)

The protein of claim 68, wherein the proliferative disease or disorder is cancer.

### Claim 70 (depends on 66, 67, 68, 69)

The protein of any of claims 66-69, wherein the gene is CBX8 or ATM.

### Claim 71 (depends on 65, 66, 67, 68, 69, 70)

The protein of any of claims 65-70, wherein the target sequence comprises a left half-site.

### Claim 72 (depends on 65, 66, 67, 68, 69, 70, 71)

The protein of any of claims 65-71, wherein the target sequence comprises a right half-site.

### Claim 73 (depends on 65, 66, 67, 68, 69, 70, 71, 72)

The protein of any of claims 65-72, wherein the target sequence comprises a left half-site and a right half-site.

### Claim 74 (depends on 71, 72, 73)

The protein of any of claims 71-73, wherein the left half-site and/or the right half-site comprise an adenine (A), a cytosine (C), or a guanine (G) at the 5′ position.

### Claim 75 (depends on 71, 72, 73)

The protein of any of claims 71-73, wherein the left half-site and/or the right half-site comprise a thymine (T) at the 5′ position.

### Claim 76 (depends on 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75)

The protein of any of claims 65-75, wherein the protein comprises an effector domain.

### Claim 77 (depends on 76)

The protein of claim 76, wherein the effector domain comprises a nuclease domain.

### Claim 78 (depends on 77)

The protein of claim 77, wherein the nuclease domain is a FokI domain.

### Claim 79 (depends on 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78)

The protein of any of claims 65-78, wherein the protein cleaves the target sequence when the protein is bound to the target sequence.

### Claim 80 (depends on 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)

The protein of any of claims 65-79, wherein the protein dimerizes to cleave the target sequence.

### Claim 81 (depends on 76)

The protein of claim 76, wherein the effector domain comprises a transcriptional activator or repressor domain, a recombinase domain, or an epigenetic modification enzyme domain.

### Claim 82 (depends on 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81)

The protein of any of claims 66-81, wherein the protein modulates transcription of the gene when the protein is bound to the target sequence.

### Claim 83 (depends on 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)

The protein of any of claims 66-82, wherein the protein increases transcription of the gene when the protein is bound to the target sequence.

### Claim 84 (depends on 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)

The protein of any of claims 66-82, wherein the protein decreases transcription of the gene when the protein is bound to the target sequence.

### Claim 85 (depends on 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)

The protein of any of claims 66-82, wherein the protein prohibits transcription of the gene when the protein is bound to the target sequence.

### Claim 86 (depends on 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85)

The protein of any of claims 65-85 wherein the genome is comprised in a cell.

### Claim 87 (depends on 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86)

The protein of any of claims 65-86 wherein the genome is comprised in a subject.

### Claim 88 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87)

A method comprising contacting a nucleic acid molecule comprising a target sequence with
(a) a protein comprising the modified TALE domain of any of claims 1-6,
(b) a protein comprising the modified TALE domain of any of claims 26-27,
(c) a protein comprising the modified TALE domain of any of claims 58-59,
(d) a protein comprising the modified TALE repeat sequence or TALE repeat array of any of claims 7-25,
(e) a protein comprising the modified TALE repeat array of any of claims 52-57,
(f) the modified TALE protein of any of claims 28-51,
(g) the modified TALE protein of any of claims 60-64, or
(h) the modified TALE protein of any of claims 65-87, under conditions suitable for the protein to bind the target sequence.

### Claim 89 (depends on 88)

The method of claim 88, wherein the contacting is in vitro.

### Claim 90 (depends on 88)

The method of claim 88, wherein the contacting is in vivo.

### Claim 91 (depends on 88, 89, 90)

The method of any of claims 88-90, wherein the nucleic acid molecule is in a cell.

### Claim 92 (depends on 88, 89, 90)

The method of any of claims 88-90, wherein the nucleic acid molecule is in a subject.

### Claim 93 (depends on 88, 89, 90, 91, 92)

The method of any of claims 88-92, wherein the nucleic acid molecule is comprised in a genome.

### Claim 94 (depends on 88, 89, 90, 91, 92, 93)

The method of any of claims 88-93, wherein the target sequence is comprised in or is in proximity to a gene known to be associated with a disease or disorder.

### Claim 95 (depends on 88, 89, 90, 91, 92, 93, 94)

The method of any of claims 88-94, wherein the target sequence is comprised in or is in proximity to a regulatory sequence of a gene known to be associated with a disease or disorder.

### Claim 96 (depends on 94, 95)

The method of claim 94 or 95, wherein the disease or disorder is a proliferative disease or disorder.

### Claim 97 (depends on 96)

The method of claim 96, wherein the proliferative disease or disorder is cancer.

### Claim 98 (depends on 94, 95, 96, 97)

The method of any of claims 94-97, wherein the gene is CBX8 or ATM.

### Claim 99 (depends on 94, 95)

The method of any of claims 94 or 95, wherein the disease or disorder is acquired immunodeficiency syndrome (AIDS), or a human immunodeficiency virus (HIV) infection.

### Claim 100 (depends on 99)

The method of claim 99, wherein the gene is CCR5.

### Claim 101 (depends on 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100)

The method of any of claims 88-100, wherein the target sequence comprises a left half-site.

### Claim 102 (depends on 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101)

The method of any of claims 88-101, wherein the target sequence comprises a right half-site.

### Claim 103 (depends on 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102)

The method of any of claims 88-102, wherein the target sequence comprises a left half-site and a right half-site.

### Claim 104 (depends on 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103)

The method of any of claims 88-103, wherein the left half-site and/or the right half-site comprise an adenine (A), a cytosine (C), or a guanine (G) at the 5′ position.

### Claim 105 (depends on 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103)

The method of any of claims 88-103, wherein the left half-site and/or the right half-site comprise a thymine (T) at the 5′ position.

### Claim 106 (depends on 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105)

The method of any of claims 88-105, wherein the protein comprises an effector domain.

### Claim 107 (depends on 106)

The method of claim 106, wherein the effector domain comprises a nuclease domain.

### Claim 108 (depends on 107)

The method of claim 107, wherein the nuclease domain is a FokI nuclease domain and/or wherein the protein is a TALEN.

### Claim 109 (depends on 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108)

The method of any of claims 88-108, wherein the protein cleaves the target sequence when the protein is bound to the target sequence.

### Claim 110 (depends on 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109)

The method of any of claims 88-109, wherein the protein dimerizes to cleave the target sequence.

### Claim 111 (depends on 106)

The method of claim 106, wherein the effector domain comprises a transcriptional activator or repressor domain, a recombinase domain, or an epigenetic modification enzyme domain.

### Claim 112 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111)

The method of any of claims 93-111, wherein the protein modulates transcription of the gene known to be associated with a disease or disorder.

### Claim 113 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112)

The method of any of claims 93-112, wherein the protein increases transcription of the gene known to be associated with a disease or disorder.

### Claim 114 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112)

The method of any of claims 93-112, wherein the protein decreases transcription of the gene known to be associated with a disease or disorder.

### Claim 115 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112)

The method of any of claims 93-112, wherein the protein prohibits transcription of the gene known to be associated with a disease or disorder.

### Claim 116 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115)

The method of any of claims 93-115, wherein the method comprises administering the protein to a subject having or diagnosed with the disease or disorder in an amount effective to ameliorate at least one symptom of the disease or disorder.

### Claim 117 (depends on 116)

The method of claim 116, wherein the disease or disorder is a proliferative disease.

### Claim 118 (depends on 116, 117)

The method of claim 116 or 117, wherein the disease or disorder is cancer.

### Claim 119 (depends on 116)

The method of claim 116, wherein the disease or disorder is acquired immunodeficiency syndrome (AIDS), or a human immunodeficiency virus (HIV) infection.

### Claim 120

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.

### Claim 121

A method of improving the specificity of a DNA-binding domain by phage-assisted, continuous evolution, 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 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.

### Claim 122 (depends on 120, 121)

The method of claim 120 or 121, wherein the positive selection construct and/or the negative selection construct is comprised on an accessory plasmid.

### Claim 123 (depends on 120, 121, 122)

The method of any one of claims 120-122, wherein the flow of host cells comprises a plurality of different negative selection constructs, wherein in each different negative selection construct, the dominant negative gene product is expressed in response to a different undesired activity of the DNA-binding domain to be evolved or an evolution product thereof.

### Claim 124 (depends on 123)

The method of claim 123, wherein the different negative selection constructs are comprised in different host cells within the flow of host cells.

### Claim 125 (depends on 120, 121, 122, 123, 124, 125)

The method of any one of claims 120-125, wherein the method further comprises (i) identifying a plurality of undesired activities of the DNA-binding domain to be evolved or an evolution product thereof, and (ii) providing a plurality of different negative selection construct selecting against different undesired activities identified in (i), wherein each different negative selection construct selects against a different undesired activity.

### Claim 126 (depends on 120, 121, 122, 123, 124, 125)

The method of any one of claims 120-125, wherein the undesired activity is DNA-binding of an off-target sequence.

### Claim 127 (depends on 124, 125, 126)

The method of any one of claims 124-126, wherein the identifying of (i) comprises performing a high-throughput screen of candidate off-target sequences.

### Claim 128 (depends on 120, 121, 122, 123, 124, 125, 126, 127)

The method of any one of claims 120-127, wherein the dominant negative gene product is expressed in response to DNA-binding of an off-target sequence by the DNA-binding domain to be evolved or an evolution product thereof.

### Claim 129 (depends on 124, 125, 126, 127, 128)

The method of any one of claims 124-128, wherein the different negative selection constructs comprise different off-target sequences.

### Claim 130 (depends on 120, 121, 122, 123, 124, 125, 126, 127, 128, 129)

The method of any one of claims 120-129, wherein the DNA-binding domain is a TALE domain.