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(12) Patent Application Publication (10) Pub. No.: US 2015/0010526 A1 Liu Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2015/0010526 A1 Liu Et Al US 2015 0010526A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0010526 A1 Liu et al. (43) Pub. Date: Jan. 8, 2015 (54) EVALUATION AND IMPROVEMENT OF (52) U.S. Cl. NUCLEASE CLEAVAGE SPECIFICITY CPC ................ CI2N 9/22 (2013.01); C12O I/6874 (2013.01); C12O I/44 (2013.01) (71) Applicant: President and Fellows of Harvard USPC ............................................ 424/94.61; 506/2 College, Cambridge, MA (US) (72) Inventors: David R. Liu, Lexington, MA (US); John Paul Guilinger, Ridgway, CO (57) ABSTRACT (US); Vikram Pattanayak, Cambridge, MA (US) Engineered nucleases (e.g., Zinc finger nucleases (ZFNs). (73) Assignee: President and Fellows of Harvard transcriptional activator-like effector nucleases (TALENs). College, Cambridge, MA (US) and others) are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of (21) Appl. No.: 14/320,271 great interest. We developed an in vitro selection method that interrogates 10' DNA sequences for their ability to be (22) Filed: Jun. 30, 2014 cleaved by active, dimeric nulceases, e.g., ZFNs and TAL O O ENs. The method revealed hundreds of thousands of DNA Related U.S. Application Data sequences, some present in the human genome, that can be (63) Continuation of application No. 14/234,031, filed on cleaved in vitro by two ZFNs, CCR5-224 and VF2468, which Mar. 24, 2014, filed as application No. PCT/US2012/ target the endogenous human CCR5 and VEGF-A genes, 047778 on Jul 22, 2012. respectively. Analysis of the identified sites in cultured (60) Provisional application No. 61/510.841, filed on Jul. human cells revealed CCR5-224-induced mutagenesis at 22, 2011. nine off-target loci. Similarly, we observed 31 off-target sites s cleaved by VF2468 in cultured human cells. Our findings Publication Classification establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN (51) Int. Cl. cleavage and suggest strategies for the improvement of future CI2N 9/22 (2006.01) nuclease design. It was also pobserved that TALENs can CI2O I/44 (2006.01) achieve cleavage specificity similar to or higher than that CI2O I/68 (2006.01) observed in ZFNs. Patent Application Publication Jan. 8, 2015 Sheet 1 of 67 US 201S/0010526 A1 S R S R 5' overhang fill-i 3 dA addition sequence adapter ligation 8888. SR 888 S Patent Application Publication Jan. 8, 2015 Sheet 2 of 67 US 201S/0010526 A1 specificity SCBE {} ,3 6 N G A T G A G G A T G A C N CCR3-224 (-) N A A A c T G c A A A A. G. N. Fig. 2A specificity Sissa WF2468 (+) N G A G T G A G G A N WF2468 (-) Patent Application Publication Jan. 8, 2015 Sheet 3 of 67 US 201S/0010526 A1 CR5-224 (+) site (-) site T C A T C C T C A T C spacer A A A C T G C A A A A G 3. - O - 8 -3.8 - 4 - 2 , 4. ,6 ,8 A specificity score Fig. 3A Patent Application Publication Jan. 8, 2015 Sheet 4 of 67 US 201S/0010526 A1 WF2A68 (--) site {-} site 5' C C C A C T C spacer G A C G C T G C 3' - O -0.8 -3.8 -.4 -0. 8.4 0.6 .8 A specificity score Fig. 3B Patent Application Publication Jan. 8, 2015 Sheet 5 of 67 US 201S/0010526 A1 {RS-234 3 station s O 9.6 - 3 titations 3 titations 4 2, 8.5 enzyme concentration in selection Fig. 4A WF468 (3% 9% 8% 7% 6% S% & statio: 4% E3 2 ?itatios 3% 3rritatics % - % 4. W 2 ry 1 nM 0.5 nM enzyme concertation in selection Fig. 4B Patent Application Publication Jan. 8, 2015 Sheet 6 of 67 US 201S/0010526 A1 *------------------------------------ Ägand#36 G-61-I Patent Application Publication Jan. 8, 2015 Sheet 7 of 67 US 201S/0010526 A1 FAG-8A standard (ng) ----------------------------------------------------4 8 12 16 20 24 . if f N. N. S S & & (ii is is is its S S C ; S.S. SoS. C a e - se 3. ww. s Fig.6A Fig.6B 43 350 : 300 3. ... 253– 15 S 300 g 150 ... Co- 0 AG Standacis 58- - AFNs - linea (FAG standards) {} } C 5 2 RS 3. rig of protei Fig. 6C Patent Application Publication Jan. 8, 2015 Sheet 8 of 67 US 201S/0010526 A1 Fig. 6D Patent Application Publication Jan. 8, 2015 Sheet 9 of 67 US 201S/0010526 A1 CRS-24 i : Fig. 7A W468 --- ke aa s s Fi 9. B Patent Application Publication Jan. 8, 2015 Sheet 10 of 67 US 2015/0010526A1 3 5 --- aw t 3 O / A\- -(- pre-Selection / /\,\, - - - - - - 4 y 2S ~A-2 ry --X - y O - K - 0.5 nM s O 2 3. 4. s 6 7 8 9 O. nun be of utations Fig. 8A -- pre-Selection ------ 4 ny - A-. nw --X - -k- (.5 v number of mutations Fig. 8B Patent Application Publication Jan. 8, 2015 Sheet 12 of 67 US 2015/0010526A1 CCR5-224 (--), 4 nM specificity SQ8 N GC. A G 3' CCR5-224 (--), M N A Arc A A C N Fig. 10A Patent Application Publication Jan. 8, 2015 Sheet 13 of 67 US 2015/0010526A1 CCR5-224 (-) S specificity SCCE N A A A C T G C A A A A G N CCFS-224 (-), M in A A Act g c A A A A G n CCR5-224 (-), 0.5 riv N A A A C T G C A A A A G N, Fig. 10B Patent Application Publication Jan. 8, 2015 Sheet 14 of 67 US 2015/0010526A1 WF2468 (--), 4 nM specificity SCO3 S WF2468 (--), 0.5 nM S’ 3. Patent Application Publication Jan. 8, 2015 Sheet 15 of 67 US 2015/0010526A1 specificity SCO8 C C G C N WF2468 (-), 0.5 nM A C C C G Fig. 10D Patent Application Publication Jan. 8, 2015 Sheet 16 of 67 US 201S/0010526 A1 Patent Application Publication Jan. 8, 2015 Sheet 17 of 67 US 201S/0010526 A1 Nºvv_2P_1>v_v_v_N" 5 - K. ovb1p9 - O - 4 Patent Application Publication Jan. 8, 2015 Sheet 18 of 67 US 201S/0010526 A1 ? Patent Application Publication Jan. 8, 2015 Sheet 19 of 67 US 2015/0010526A1 WF2468 (--), 4 nM WF2468 (-), 4 nM G C A s N specificity WF2468 (--), when f2468 (-), whes (3-3G is fatated s T G G : C C A : A N G A G T G A G G A N. N G A C G C T G C T N. WF2463 (+), when {-}{ is nutated VF2468 (-), when {-}G is mutated x f x C C A A. N 5' s S 3' WF2468 (+), WF2468 (-), wher. --G and {-}G are notated when {{-}G and (-)G are nutated & are erger N G A G T G A G G A N, S 3 Fig. 13 Patent Application Publication Jan. 8, 2015 Sheet 20 of 67 US 2015/0010526A1 3. s ; : is a is is a & a 2. 22 2.wo a.a. F. a 2. s 2 : a 2.2 S 2 : a 2 w s a 2. 2. 8 . (- - a. c. - - 8. s ; : a ; : ee a a - - c tr i. i. if awww.www.www.www.www. 22 P ef s r is 2 : 22. e a 2. s a 2. s 22 s a a r R na n O a 2 a a i 3 w a a a 2. a ................ 5. d. ... c. c. 8 B 3 S. a a Ei & 2 2. 22. es 2 : s 22re &s isna a 2. 2 : a 2. 2. ' a 2 : a 2. a 2. w 2 2 w it. ... ... - - . ... Patent Application Publication Jan. 8, 2015 Sheet 21 of 67 US 2015/0010526A1 s: a s a i. is 3 a 2. 2. a. 2 : iss ? Ss a a 2 2 a 2 : ...Y 2. wat a 22. ... - a. c. ... - -, is. a. : & iss ; : s:s i. 2, ase Ca 22. 2 s s 2 : a m s e e 22 s 2 2. s a a : 3 ra. O e a a sess e - 22 a la a a 2. at a 3. A- A. a. c. C. C.- 3. - - i.- i. ;- : a E. is ; : Ea a 2. 2 s fi- fif , , , , , tea 2 : Ya. - 2. ra 2 : ran 2. 2. a 2 : a 2 : a 2 2. a 2. 22 2. Aw C. C. i. e. - C. h. Patent Application Publication Jan. 8, 2015 Sheet 22 of 67 US 201S/0010526 A1 O% 45% si. 35% CCR5-224, , , 3% 7 5% 20% 3 6 S. S 5 O% 3. ce S96 Q 4 4 5 6 7 spacer ength Fig. 15A CCRS-224, 7 is 6 - s to a 5 6 spacer lengt; Fig. 15B Patent Application Publication Jan. 8, 2015 Sheet 23 of 67 US 2015/0010526A1 CCR5-224, 2 7 3 & 5. s . S. O a s 6 Spacer ength Fig. 15C CCR5-224, 4 5. s 6 5 to spacer length Fig. 15D Patent Application Publication Jan. 8, 2015 Sheet 24 of 67 US 2015/0010526A1 WF2468, O.S. -- :-- 7 s s . c s C sa > & spacer length Fig. 16A WF2468, in 67 s 4. spacer length Fig. 16B Patent Application Publication Jan. 8, 2015 Sheet 25 of 67 US 2015/0010526A1 WF2468, 7 6 ma 4. A. S 6 7 O% Spacer ength Fig. 16C 5% AS.6 a0% 3596. WF468, 4 ti 3% 2% O% 56f --ra S96 O% 4.
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