FUSION MOLECULES OF RATIONALLY-DESIGNED DNA-BINDING PROTEINS AND EFFECTOR DOMAINS

• US 20110123509A1
• Filed: 10/28/2010
• Published: 05/26/2011
• Est. Priority Date: 04/28/2008
• Status: Abandoned Application

• Current Assignee: Precision BioSciences Incorporated
• Original Assignee: Precision BioSciences Incorporated
• Inventors: JANTZ, Derek, NICHOLSON, Michael G., SMITH, James J.
• Application Number:
  US12/914,014
• Publication Number:
  US 20110123509A1
• Time in Patent Office: Days
• US Class Current: 424/94.3
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Targeted transcriptional effectors (transcription activators and transcription repressors) derived from meganucleases are described. Also described are nucleic acids encoding same, and methods of using same to regulate gene expression. The targeted transcriptional effectors can comprise (i) a meganuclease DNA-binding domain lacking endonuclease cleavage activity that binds to a target recognition site; and (ii) a transcription effector domain.

Claims

1. A targeted transcriptional effector comprising:

(i) an inactive meganuclease DNA-binding domain that binds to a target recognition site; and

(ii) a transcription effector domain, wherein binding of the meganuclease DNA-binding domain targets the transcriptional effector to a gene of interest.

2. The targeted transcriptional effector of claim 1, further comprising a domain linker joining the meganuclease DNA-binding domain and the transcription effector domain.

3. The targeted transcriptional effector of claim 2, wherein the domain linker comprises a polypeptide.

4. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain is altered from a naturally-occurring meganuclease by at least one point mutation which reduces or abolishes endonuclease cleavage activity.

5. The targeted transcriptional effector of claim 1, further comprising a nuclear localization signal.

6. The method of claim 1, wherein the transcriptional effector domain is a transcription activator.

7. The method of claim 1, wherein the transcriptional effector domain is a transcription repressor.

8. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered specificity for at least one recognition sequence half-site relative to a wild-type I-CreI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1; and

having specificity for a recognition sequence half-site which differs by at least one base pair from a half-site within an I-CreI meganuclease recognition sequence selected from the group consisting of SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4 and SEQ ID NO;

5;

wherein said recombinant meganuclease comprises at least one modification of Table 1 and a modification which reduces or abolishes said endonuclease cleavage activity.

9. The targeted transcriptional effector of claim 8, wherein the modification which reduces or abolishes said endonuclease cleavage activity is Q47E.

10. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered specificity for at least one recognition sequence half-site relative to a wild-type I-MsoI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 6-160 of the I-MsoI meganuclease of SEQ ID NO;

6; and

having specificity for a recognition sequence half-site which differs by at least one base pair from a half-site within an I-MsoI meganuclease recognition sequence selected from the group consisting of SEQ ID NO;

7 and SEQ ID NO;

8;

wherein said recombinant meganuclease comprises at least one modification of Table 2 and a modification which reduces or abolishes said endonuclease cleavage activity.

11. The targeted transcriptional effector of claim 10, wherein the modification which reduces or abolishes said endonuclease cleavage activity is D22N.

12. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered specificity for a recognition sequence relative to a wild-type 1-SceI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 3-186 of the I-SceI meganuclease of SEQ ID NO;

9; and

having specificity for a recognition sequence which differs by at least one base pair from an I-SceI meganuclease recognition sequence of SEQ ID NO;

10 and SEQ ID NO;

11;

wherein said recombinant meganuclease comprises at least one modification of Table 3 and a modification which reduces or abolishes said endonuclease cleavage activity.

13. The targeted transcriptional effector of claim 12, wherein the modification which reduces or abolishes said endonuclease cleavage activity is D44N or D145N.

14. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered specificity for at least one recognition sequence half-site relative to a wild-type I-Ceul meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 5-211 of the I-CeuI meganuclease of SEQ ID NO;

12; and

having specificity for a recognition sequence half-site which differs by at least one base pair from a half-site within an I-CeuI meganuclease recognition sequence selected from the group consisting of SEQ ID NO;

13 and SEQ ID NO;

14;

wherein said recombinant meganuclease comprises at least one modification of Table 4 and a modification which reduces or abolishes said endonuclease cleavage activity.

15. The targeted transcriptional effector of claim 14, wherein the modification which reduces or abolishes said endonuclease cleavage activity is E66Q.

16. The targeted transcriptional effector of claim 1 wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered specificity for at least one recognition sequence half-site relative to a wild-type I-CreI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1; and

having specificity for a recognition sequence half-site which differs by at least one base pair from a half-site within an I-CreI meganuclease recognition sequence selected from the group consisting of SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4 and SEQ ID NO;

5;

wherein;

(1) specificity at position −

1 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of Q70, C70, L70, Y75, Q75, H75, H139, Q46 and H46;

(b) to an A on a sense strand by a modification selected from the group consisting of Y75, L75, C75, Y139, C46 and A46;

(c) to a G on a sense strand by a modification selected from the group consisting of K70, E70, E75, E46 and D46;

(d) to a C on a sense strand by a modification selected from the group consisting of H75, R75, H46, K46 and R46;

or

(e) to any base on a sense strand by a modification selected from the group consisting of G70, A70, S70 and G46; and

/or

(2) specificity at position −

2 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of Q70, T44, A44, V44, I44, L44, and N44;

(b) to a C on a sense strand by a modification selected from the group consisting of E70, D70, K44 and R44;

(c) to a G on a sense strand by a modification selected from the group consisting of H70, D44 and E44;

or

(d) to an A or T on a sense strand by a modification comprising C44; and

/or

(3) specificity at position −

3 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of Q68 and C24;

(b) to a C on a sense strand by a modification selected from the group consisting of E68, F68, K24 and R24;

(c) to a T on a sense strand by a modification selected from the group consisting of M68, C68, L68 and F68;

(d) to an A or C on a sense strand by a modification comprising H68;

(e) to a C or T on a sense strand by a modification comprising Y68;

or

(f) to a G or T on a sense strand by a modification comprising K68; and

/or

(4) specificity at position −

4 has been altered;

(a) to a C on a sense strand by a modification selected from the group consisting of E77 and K26;

(b) to a G on a sense strand by a modification selected from the group consisting of E26 and R77;

(c) to a C or T on a sense strand by a modification comprising S77;

or

(d) to a any base on a sense strand by a modification comprising S26; and

/or

(5) specificity at position −

5 has been altered;

(a) to a C on a sense strand by a modification comprising E42;

(b) to a G on a sense strand by a modification comprising R42;

(c) to an A or G on a sense strand by a modification selected from the group consisting of C28 and Q42;

or

(d) to any base on a sense strand by a modification of selected from the group consisting of M66 and K66; and

/or

(6) specificity at position −

6 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of C40, 140, V40, C79, I79, V79, and Q28;

(b) to a C on a sense strand by a modification selected from the group consisting of E40 and R28;

or

(c) to a G on a sense strand by a modification comprising R40; and

/or

(7) specificity at position −

7 has been altered;

(a) to a C on a sense strand by a modification selected from the group consisting of E38, K30 and R30;

(b) to a G on a sense strand by a modification selected from the group consisting of K38, R38 and E30;

(c) to a T on a sense strand by a modification selected from the group consisting of 138 and L38;

or

(d) to an A or G on a sense strand by a modification comprising C38;

or

(e) to any base on a sense strand by a modification selected from the group consisting of H38, N38 and Q30; and

/or

(8) specificity at position −

8 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of L33, V33, I33, F33 and C33;

(b) to a C on a sense strand by a modification selected from the group consisting of E33 and D33;

(c) to a G on a sense strand by a modification consisting of K33;

(d) to an A or C on a sense strand by a modification comprising R32;

or

(e) to an A or G on a sense strand by a modification comprising R33; and

/or

(9) specificity at position −

9 has been altered;

(a) to a C on a sense strand by a modification comprising E32;

(b) to a G on a sense strand by a modification selected from the group consisting of R32 and K32;

(c) to a T on a sense strand by a modification selected from the group consisting of L32, V32, A32 and C32;

(d) to a C or T on a sense strand by a modification selected from the group consisting of D32 and 132;

or

(e) to any base on a sense strand by a modification selected from the group consisting of S32, N32, H32, Q32 and T32.

17. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered specificity for at least one recognition sequence half-site relative to a wild-type I-MsoI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 6-160 of the I-MsoI meganuclease of SEQ ID NO;

6; and

having specificity for a recognition sequence half-site which differs by at least one base pair from a half-site within an I-MsoI meganuclease recognition sequence selected from the group consisting of SEQ ID NO;

7 and SEQ ID NO;

8;

wherein;

(1) specificity at position −

1 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of K75, Q77, A49, C49 and K79;

(b) to a T on a sense strand by a modification selected from the group consisting of C77, L77 and Q79;

or

(c) to a G on a sense strand by a modification selected from the group consisting of K77, R77, E49 and E79; and

/or

(2) specificity at position −

2 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of Q75, K81, C47, I47 and L47;

(b) to a C on a sense strand by a modification selected from the group consisting of E75, D75, R47, K47, K81 and R81;

or

(c) to a G on a sense strand by a modification selected from the group consisting of K75, E47 and E81; and

/or

(3) specificity at position −

3 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of Q72, C26, L26, V26, A26 and 126;

(b) to a C on a sense strand by a modification selected from the group consisting of E72, Y72, H26, K26 and R26;

or

(c) to a T on a sense strand by a modification selected from the group consisting of K72, Y72 and H26; and

/or

(4) specificity at position −

4 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of K28, K83 and Q28;

(b) to a G on a sense strand by a modification selected from the group consisting of R83 and K83;

or

(c) to an A on a sense strand by a modification selected from the group consisting of K28 and Q83; and

/or

(5) specificity at position −

5 has been altered;

(a) to a G on a sense strand by a modification selected from the group consisting of R45 and E28;

(b) to a T on a sense strand by a modification comprising Q28;

or

(c) to a C on a sense strand by a modification comprising R28; and

/or

(6) specificity at position −

6 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of K43, V85, L85 and Q30;

(b) to a C on a sense strand by a modification selected from the group consisting of E43, E85, K30 and R30;

or

(c) to a G on a sense strand by a modification selected from the group consisting of R43, K43, K85, R85, E30 and D30; and

/or

(7) specificity at position −

7 has been altered;

(a) to a C on a sense strand by a modification selected from the group consisting of E32 and E41;

(b) to a G on a sense strand by a modification selected from the group consisting of R32, R41 and K41;

(c) to a T on a sense strand by a modification selected from the group consisting of K32, M41, L41 and I41; and

/or

(8) specificity at position −

8 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of K32 and 1(35;

(b) to a C on a sense strand by a modification comprising E32;

or

(c) to a G on a sense strand by a modification consisting of K32, K35 and R35; and

/or

(9) specificity at position −

9 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of N34 and H34;

(b) to a T on a sense strand by a modification selected from the group consisting of S34, C34, V34, T34 and A34;

or

(c) to a G on a sense strand by a modification selected from the group consisting of K34, R34 and H34.

18. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises recombinant meganuclease having altered specificity for a recognition sequence relative to a wild-type I-SceI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 3-186 of the I-SceI meganuclease of SEQ ID NO;

9; and

having specificity for a recognition sequence which differs by at least one base pair from an I-SceI meganuclease recognition sequence of SEQ ID NO;

10 and SEQ ID NO;

11;

wherein;

(1) specificity at position 4 has been altered;

(a) to an A on a sense strand by a modification comprising K₅₀;

(b) to a T on a sense strand by a modification selected from the group consisting of K57, M57 and Q50;

or

(c) to a G on a sense strand by a modification selected from the group consisting of E50, R57 and K57; and

/or

(2) specificity at position 5 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of K48, Q102;

(b) to a G on a sense strand by a modification selected from the group consisting of E48, K102 and R102;

or

(c) to a T on a sense strand by a modification selected from the group consisting of Q48, C 102, L102 and V102; and

/or

(3) specificity at position 6 has been altered;

(a) to an A on a sense strand by a modification comprising K59;

(b) to a C on a sense strand by a modification selected from the group consisting of R59 and K59;

or

(c) to a G on a sense strand by a modification selected from the group consisting of K84 and E59; and

/or

(4) specificity at position 7 has been altered;

(a) to a C on a sense strand by a modification selected from the group consisting of R46, K46 and E86;

(b) to a G on a sense strand by a modification selected from the group consisting of K86, R86 and E46;

or

(c) to an A on a sense strand by a modification selected from the group consisting of C46, L46 and V46; and

/or

(5) specificity at position 8 has been altered;

(a) to a C on a sense strand by a modification selected from the group consisting of E88, R61 and H61;

(b) to a T on a sense strand by a modification selected from the group consisting of K88, Q61 and H61;

or

(c) to an A on a sense strand by a modification selected from the group consisting of K61, S61, V61, A61 and L61; and

/or

(6) specificity at position 9 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of C98, V98 and L98;

(b) to a C on a sense strand by a modification selected from the group consisting of R98 and K98;

or

(c) to a G on a sense strand by a modification selected from the group consisting of E98 and D98; and

/or

(7) specificity at position 10 has been altered;

(a) to a C on a sense strand by a modification selected from the group consisting of K96 and R96;

(b) to a G on a sense strand by a modification selected from the group consisting of D96 and E96;

or

(c) to an A on a sense strand by a modification selected from the group consisting of C96 and A96; and

/or

(8) specificity at position 11 has been altered;

(a) to a T on a sense strand by a modification comprising Q90;

(b) to a C on a sense strand by a modification selected from the group consisting of K90 and R90;

or

(c) to a G on a sense strand by a modification comprising E90; and

/or

(9) specificity at position 12 has been altered;

(a) to an A on a sense strand by a modification comprising Q193;

(b) to a C on a sense strand by a modification selected from the group consisting of E165, E193 and D193;

or

(c) to a G on a sense strand by a modification selected from the group consisting of K165 and R165; and

/or

(10) specificity at position 13 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of Q193, C163 and L163;

(b) to a G on a sense strand by a modification selected from the group consisting of E193, D193, K163 and R192;

or

(c) to an A on a sense strand by a modification selected from the group consisting of C193 and L193; and

/or

(11) specificity at position 14 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of K161 and Q192;

(b) to an A on a sense strand by a modification selected from the group consisting of L192 and C192;

(c) to a G on a sense strand by a modification selected from the group consisting of K147, K161, R161, R197, D192 and E192;

or

(d) to a T on a sense strand by a modification selected from the group consisting of K161 and Q192; and

/or

(12) specificity at position 15 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of C151, L151 and K151;

(b) to a G on a sense strand by a modification comprising K₁₅₁;

or

(c) to a C on a sense strand by a modification comprising E 151; and

/or

(13) specificity at position 17 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of G152 and Q150;

(b) to a C on a sense strand by a modification selected from the group consisting of K152 and K150;

or

(c) to a G on a sense strand by a modification selected from the group consisting of N152, 5152, D152, D150 and E150; and

/or

(14) specificity at position 18 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of H155 and Y155;

(b) to a C on a sense strand by a modification selected from the group consisting of R155 and K155;

or

(c) to an A on a sense strand by a modification selected from the group consisting of K155 and C155.

19. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered specificity for at least one recognition sequence half-site relative to a wild-type I-CeuI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 5-211 of the I-CeuI meganuclease of SEQ ID NO;

12; and

having specificity for a recognition sequence half-site which differs by at least one base pair from a half-site within an I-CeuI meganuclease recognition sequence selected from the group consisting of SEQ ID NO;

13 and SEQ ID NO;

14;

wherein;

(1) specificity at position −

1 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of C92, A92 and V92;

(b) to a T on a sense strand by a modification selected from the group consisting of Q116 and Q92;

or

(c) to a G on a sense strand by a modification selected from the group consisting of E116 and E92; and

/or

(2) specificity at position −

2 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of Q117, C90, L90 and V90;

(b) to a G on a sense strand by a modification selected from the group consisting of K117, R124, K124, E124, E90 and D90;

or

(c) to a C on a sense strand by a modification selected from the group consisting of E117, D117, R174, K124, K90, R90 and K68; and

/or

(3) specificity at position −

3 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of C70, V70, T70, L70 and K70;

(b) to a T on a sense strand by a modification comprising Q70;

(b) to a C on a sense strand by a modification consisting of K70; and

/or

(4) specificity at position −

4 has been altered;

(a) to a C on a sense strand by a modification selected from the group consisting of E126, D126, R88, K88 and K72;

(b) to a T on a sense strand by a modification selected from the group consisting of K126, L126 and Q88;

or

(c) to an A on a sense strand by a modification selected from the group consisting of Q126, N126, K88, L88, C88, C72, L72 and V72; and

/or

(5) specificity at position −

5 has been altered;

(a) to a G on a sense strand by a modification selected from the group consisting of E74, K128, R128 and E128;

(b) to a T on a sense strand by a modification selected from the group consisting of C128, L128, V128 and T128;

or

(c) to an A on a sense strand by a modification selected from the group consisting of C74, L74, V74 and T74; and

/or

(6) specificity at position −

6 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of K86, C86 and L86;

(b) to a C on a sense strand by a modification selected from the group consisting of D86, E86, R84 and K84;

or

(c) to a G on a sense strand by a modification selected from the group consisting of K128, R128, R86, K86 and E84; and

/or

(7) specificity at position −

7 has been altered;

(a) to a C on a sense strand by a modification selected from the group consisting of R76, K76 and H76;

(b) to a G on a sense strand by a modification selected from the group consisting of E76 and R84;

or

(c) to a T on a sense strand by a modification consisting of H76 and Q76; and

/or

(8) specificity at position −

8 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of Y79, R79 and Q76;

(b) to a C on a sense strand by a modification selected from the group consisting of D79, E79, D76 and E76;

or

(c) to a G on a sense strand by a modification selected from the group consisting of R79, K79, K76 and R76; and

/or

(9) specificity at position −

9 has been altered;

(a) to a T on a sense strand by a modification selected from the group consisting of K78, V78, L78, C78 and T78;

(b) to a C on a sense strand by a modification selected from the group consisting of D78 and E78;

or

(c) to a G on a sense strand by a modification selected from the group consisting of R78, K78 and H78.

20. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-CreI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1;

wherein DNA-binding affinity has been increased by at least one modification corresponding to;

(a) substitution of E80, D137, I81, L112, P29, V64 or Y66 with H, N, Q, S, T, K or R;

or

(b) substitution of T46, T140 or T143 with K or R.

21. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-CreI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1;

wherein DNA-binding affinity has been decreased by at least one modification corresponding to;

(a) substitution of K34, K48, R51, K82, K116 or K139 with H, N, Q, S, T, D or E;

or

(b) substitution of I81, L112, P29, V64, Y66, T46, T140 or T143 with D or E.

22. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-MsoI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 6-160 of the I-MsoI meganuclease of SEQ ID NO;

6;

wherein DNA-binding affinity has been increased by at least one modification corresponding to;

(a) substitution of E147, 185, G86 or Y118 with H, N, Q, S, T, K or R;

or

(b) substitution of Q41, N70, S87, T88, H89, Q122, Q139, S150 or N152 with K or R.

23. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-MsoI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 6-160 of the I-MsoI meganuclease of SEQ ID NO;

6;

wherein DNA-binding affinity has been decreased by at least one modification corresponding to;

(a) substitution of K36, R51, K123, K143 or R144 with H, N, Q, S, T, D or E;

or

(b) substitution of 185, G86, Y118, Q41, N70, S87, T88, H89, Q122, Q139, S150 or N152 with D or E.

24. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-SceI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 3-186 of the I-SceI meganuclease of SEQ ID NO;

9;

wherein DNA-binding affinity has been increased by at least one modification corresponding to;

(a) substitution of D201, L19, L80, L92, Y151, Y188, 1191, Y199 or Y222 with H, N, Q, S, T, K or R;

or

(b) substitution of N15, N17, S81, H84, N94, N120, T156, N157, S159, N163, Q165, S166, N194 or 5202 with K or R.

25. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-SceI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 3-186 of the I-SceI meganuclease of SEQ ID NO;

9;

wherein DNA-binding affinity has been decreased by at least one modification corresponding to;

(a) substitution of K20, K23, K63, K122, K148, K153, K190, K193, K195 or K223 with H, N, Q, S, T, D or E;

or

(b) substitution of L19, L80, L92, Y151, Y188, 1191, Y199, Y222, N15, N17, S81, H84, N94, N120, T156, N157, S159, N163, Q165, S166, N194 or S202 with D or E.

26. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-CeuI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 5-211 of the I-CeuI meganuclease of SEQ ID NO;

12;

wherein DNA-binding affinity has been increased by at least one modification corresponding to;

(a) substitution of D25 or D128 with H, N, Q, S, T, K or R;

or

(b) substitution of S68, N70, H94, S117, N120, N129 or H172 with K or R.

27. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-CeuI meganuclease, comprising:

a polypeptide having at least 85% sequence similarity to residues 5-211 of the I-CeuI meganuclease of SEQ ID NO;

12;

wherein DNA-binding affinity has been decreased by at least one modification corresponding to;

(a) substitution of K21, K28, K31, R112, R114 or R130 with H, N, Q, S, T, D or E;

or

(b) substitution of S68, N70, H94, S117, N120, N129 or H172 with D or E.

28. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease monomer having altered affinity for dimer formation with a reference meganuclease monomer, comprising:

a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1;

wherein affinity for dimer formation has been altered by at least one modification corresponding to;

(a) substitution of K7, K57 or K96 with D or E;

or

(b) substitution of E8 or E61 with K or R.

29. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease heterodimer comprising:

a first polypeptide having at least 85% sequence similarity to residues 2-153 of the 1-CreI meganuclease of SEQ ID NO;

1;

wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution of K7, K57 or K96 with D or E; and

a second polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1;

wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution of E8 or E61 with K or R.

30. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease monomer having altered affinity for dimer formation with a reference meganuclease monomer, comprising:

a polypeptide having at least 85% sequence similarity to residues 6-160 of the I-MsoI meganuclease of SEQ ID NO;

6;

wherein affinity for dimer formation has been altered by at least one modification corresponding to;

(a) substitution of R302 with D or E;

or

(b) substitution of D20, E11 or Q64 with K or R.

31. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease heterodimer comprising:

a first polypeptide having at least 85% sequence similarity to residues 6-160 of the I-MsoI meganuclease of SEQ ID NO;

6;

wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution of R302 with D or E; and

a second polypeptide having at least 85% sequence similarity to residues 6-160 of the I-MsoI meganuclease of SEQ ID NO;

6;

wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution of D20, E11 or Q64 with K or R.

32. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease monomer having altered affinity for dimer formation with a reference meganuclease monomer, comprising:

a polypeptide having at least 85% sequence similarity to residues 5-211 of the I-CeuI meganuclease of SEQ ID NO;

12;

wherein affinity for dimer formation has been altered by at least one modification corresponding to;

(a) substitution of R93 with D or E;

or

(b) substitution of E152 with K or R.

33. The targeted transcriptional effector of claim 1, wherein the meganuclease DNA-binding domain comprises a recombinant meganuclease heterodimer comprising:

a first polypeptide having at least 85% sequence similarity to residues 5-211 of the I-CeuI meganuclease of SEQ ID NO;

12;

wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution of R93 with D or E; and

a second polypeptide having at least 85% sequence similarity to residues 5-211 of the I-CeuI meganuclease of SEQ ID NO;

12;

wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution of E152 with K or R.

34. The targeted transcriptional effector of claim 1, wherein the recombinant meganuclease monomer or heterodimer further comprises at least one modification selected from Table 1.

35. A nucleic acid encoding the targeted transcriptional effector of claim 1.

36. A method for treating a disease or condition in a subject in need thereof, the method comprising:

introducing the nucleic acid of claim 35 into a subject, whereby the polypeptide encoded by the nucleic acid binds to the target site and affects transcription of the gene of interest.

37. A method for treating a disease or condition in a subject in need thereof, the method comprising:

introducing the targeted transcriptional effector of claim 1 into a subject, whereby the polypeptide binds to the target site and affects transcription of the gene of interest.