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United States Patent (10) Patent No.: US 9,359,599 B2 Liu Et Al US00935.9599B2 (12) United States Patent (10) Patent No.: US 9,359,599 B2 Liu et al. (45) Date of Patent: Jun. 7, 2016 (54) ENGINEERED TRANSCRIPTION 8,759,104 B2 6/2014 Unciti-Broceta et al. ACTIVATOR-LIKE EFFECTOR (TALE) 76. R: 2.38 Eas st al DOMAINS AND USES THEREOF 8,846,578- W B2 9/2014 McCraya ca. et al. 2006, OO88864 A1 4/2006 Smoke et al. (71) Applicant: President and Fellows of Harvard 2008. O1822.54 A1 T/2008 R al College, Cambridge, MA (US) 2009,0130718 A1 5, 2009 Short 2009, 0234109 A1 9, 2009 Han et al. (72) Inventors: David R. Liu, Lexington, MA (US); 2010/0076057 A1 3/2010 Sontheimer et al. John Paul Guili Rid CO 2011 0104787 A1 5, 2011 Church et al. onn raui Gunger, Kigway, , 2011/O189776 A1 8/2011 Terns et al. (US); Vikram Pattanayak, Cambridge, 2012/0141523 A1 6, 2012 Castado et al. MA (US) 2012/0270273 Al 10/2012 Zhang et al. 2013/01 17869 A1* 5, 2013 Duchateau ............... C12N 9/22 (73) Assignee: President and Fellows of Harvard 2013,0130248 A1 5, 2013 H itz et al 800, 13 College, Cambridge, MA (US) 2013/0344117 A1 12/2013 MirosevichaWa ca. et al. 2014,0005269 A1 1, 2014 N luka et al. (*) Notice: Subject to any disclaimer, the term of this 2014, OO18404 A1 1, 2014 E. e al patent is extended or adjusted under 35 2014/0044793 A1 2/2014 GO11 et al. U.S.C. 154(b) by 0 days. 2014/0068797 A1 3/2014 Doudna et al. 2014/O127752 A1 5, 2014 Zhou et al. 2014/O141094 A1 5/2014 Smyth et al. (21) Appl. No.: 14/320,519 2014/0141487 A1 5/2014 Feldman et al. 2014/O186843 A1 7/2014 Zhang9. et al. (22) Filed: Jun. 30, 2014 2014/0186958 A1 7/2014 Zhang et al. (65) Prior Publication Data (Continued) US 2015/0056177 A1 Feb. 26, 2015 FOREIGN PATENT DOCUMENTS O O AU 2012244264 11, 2012 Related U.S. Application Data CN 103233028 A 8, 2013 CN 103388006 A 11, 2013 (60) Provisional application No. 61/868,846, filed on Aug. CN 103614415 A 3, 2014 22, 2013. CN 103.642836 A 3, 2014 (51) Int. Cl. (Continued) CI2N I5/0 (2006.01) CI2N 9/22 (2006.01) OTHER PUBLICATIONS (52) U.S. Cl. International Search Report and Written Opinion for PCT/US2014/ CPC CI2N 15/01 (2013.01); C12N 9/22 (2013.01); O52231, mailed Dec. 4, 2014 C07K 2319/00 (2013.01); C07K 2319/80 • I (201 3.01 ) (Continued) (58) Field of Classification Search None See application file for complete search history. Primary Examiner — Suzanne M Noakes (56) Ref Cited Assistant Examiner — Jae W Lee eerees e (74) Attorney, Agent, or Firm — Wolf, Greenfield & Sacks, U.S. PATENT DOCUMENTS P.C. 5,780,053 A 7/1998 Ashley et al. 6,057,153 A 5/2000 George et al. (57) ABSTRACT 6,453,242 B1 9/2002 Eisenberg et al. 6,503,717 B2 1/2003 Case et al. 6,534,261 B1 3/2003 Cox, III et al. Engineered transcriptional activator-like effectors (TALEs) 6,599,692 B1 7/2003 Case et al. are versatile tools for genome manipulation with applications 6,607,882 B1 8/2003 Cox, III et al. in research and clinical contexts. One current drawback of 6,824,978 B1 1 1/2004 Cox, III et al. 6,933,113 B2 8, 2005 Case et al. TALEs is their tendency to bind and cleave off-target 6,979,539 B2 12/2005 Cox, III et al. sequence, which hampers their clinical application and ren 7,013,219 B2 3, 2006 Case et al. ders applications requiring high-fidelity binding unfeasible. 7,163,824 B2 1/2007 Cox, III et al. This disclosure provides engineered TALE domains and 7,479,573 B2 1/2009 Chu et al. 7,794,931 B2 9, 2010 Breaker et al. TALEs comprising Such engineered domains, e.g., TALE 8,569.256 B2 10/2013 Heyes et al. nucleases (TALENs). TALE transcriptional activators, TALE 8,680,069 B2 3/2014 de Fougerolles et al. transcriptional repressors, and TALE epigenetic modification 8,691,750 B2 4/2014 Constien et al. enzymes, with improved specificity and methods for gener 8,709,466 B2 4/2014 Coady et al. 8,728,526 B2 5, 2014 Heller ating and using such TALEs. 8,748,667 B2 6, 2014 Budzik et al. 8,758,810 B2 6, 2014 Okada et al. 8,759,103 B2 6, 2014 Kim et al. 28 Claims, 34 Drawing Sheets US 9,359,599 B2 Page 2 (56) References Cited WO WO 2011/O17293 A2 2/2011 WO WO 2011/O53982 A2 5, 2011 U.S. PATENT DOCUMENTS WO WO 2012/054726 A1 4, 2012 WO WO 2012/065043 A2 5, 2012 2014/0234289 A1 8, 2014 Liu et all WO WO 2012/138927 A2 10, 2012 2014/0273O37 A1 9, 2014 Wu WO WO 2012/158985 A2 11/2012 2014/0273226 A1 9, 2014 Wu WO WO 2012/158986 A2 11/2012 2014/0273230 A1 9, 2014 Chen et al. WO WO 2012, 164565 A1 12/2012 2014/0295556 Al 10/2014 Joung et al. WO WO 2013/O12674 A1 1/2013 2014/0295557 A1 10/2014 Joung et al. 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