SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLEMENTARY INFORMATION doi: /nture10177 MDYKDHDGDYKDHDIDYKDD DDKMAPKKKRKVGIHGVPAA MAERPFQCRICMRKFAQSGD LTRHTKIHTGEKPFQCRICM RNFSRSDVLSEHIRTHTGEK PFACDICGKKFADRSNRIKH TKIHTGSQKPFQCRICMRNF SRSDNLSEHIRTHTGEKPFA CDICGRKFAQNATRINHTKI HLRGSQLVKSELEEKKSELR HKLKYVPHEYIELIEIARNS TQDRILEMKVMEFFMKVYGY RGKHLGGSRKPDGAIYTVGS PIDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKH INPNEWWKVYPSSVTEFKFL FVSGHFKGNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMI KAGTLTLEEVRRKFNNGEIN F MDYKDHDGDYKDHDIDYKDD DDKMAPKKKRKVGIHGVPER PFQCQICMRNFSRSDSLSVH IRTHTGEKPFACDICGRKFA TSGHLSRHTKIHTGSQKPFQ CRICMRNFSRSDHLSQHIRT HTGEKPFACDICGRKFAHAS TRHCHTKIHLRGSQLVKSEL EEKKSELRHKLKYVPHEYIE LIEIARNSTQDRILEMKVME FFMKVYGYRGKHLGGSRKPD GAIYTVGSPIDYGVIVDTKA YSGGYNLPIGQADEMQRYVE ENQTRNKHINPNEWWKVYPS SVTEFKFLFVSGHFKGNYKA QLTRLNHITNCNGAVLSVEE LLIGGEMIKAGTLTLEEVRR KFNNGEINF Supplementry Figure 1 - F9 ZFN pir mino cid sequence Amino cid sequence of FLAG-tgged, left F9 ZFN nd, right F9 ZFN. 1

2 RESEARCH SUPPLEMENTARY INFORMATION - hf9 ZFN Donor - ZFN Undigested Cel-I nuclese clevge Undigested 0 17 % Indels NheI clevge % HDR Supplementry Figure 2 - F9 ZFNs cleve humn F9 intron 1 nd induce homologydirected repir in Hep3B cells., Trnsfection of 400 ng of ZFN expression plsmid into Hep3B cells results in clevge of the humn F9 intron 1 t dy 3 post-trnsfection. Cel-1 ssy performed with PCR using 32 P-leled nucleotides, followed y PAGE nd nd intensity quntifiction y utordiogrphy., Co-trnsfection of 400 ng of ZFN expression plsmid with incresing mounts of NheI donor plsmid (0, 1, 2, nd 4 μg) results in incresing levels of HDR t dys 3 post-trnsfection, wheres trnsfection of the NheI donor lone (4 μg) does not result in detectle HDR. HDR PCR performed with PCR using 32 P-leled nucleotides, followed y PAGE nd nd intensity quntifiction y utordiogrphy. Lnes with no quntifiction hd no detectle HDR. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Supplementry Figure 3 AAAGCTGACTGGCCCTGGTGCCAGGTACTGTGTCAGGGCACTAGGGGTATGGGGACAGGTTAGTCCACC AAAGCTGACTGGCCCTGGTGCCAGGTACTGTGTCATGGTACTAGGGGTATGGGGACAGGTTAGTCCACC AAAGCTGACTGGCCCTGGTGCCAGGTACTGTGTCAGGTTACTAGGGGTATGGGGACAGGTTAGTCCACC AAAGCTGACTGGCCCTGGTGCCAGGTACTGTGTCAGGTTACTAGGGGTATGGGGACAGGTTAGTCCACC CTGGTGCCAGGTACTGTGTCAGGGAGGCCTCAGTGAGGA1GTGGCCAAAGGTTACTAGGGGTATGGGGAC CTGGTGCCAGGTACTGTGTCAGGGAGGCCTCAGTGAGGA1GTGGCCAAAGGTTACTAGGGGTATGGGGAC CTGGTGCCAGGTACTGTGTCAGGGAGGCCTCAGTGAGGA1GTGGCCAAAGGGTACTAGGGGTATGGGGAC CTGGTGCCAGGTACTGTGTCAGGGAGGCCTCAGTGAGGA1GTGGSCCAAGGGTACTAGGGGTATGGGGAC CTGGTGCCAGGTACTGTGTCAGGGAGGCCTCAGTGAGGA1GTGGCCAAAGGGTACTAGGGGTATGGGGAC CTGGTGCCAGGTACTGTGTCAGGGAGGCCTCAGTGAGG1AGTGGCCAAAGGGTACTAGGGGTATGGGGAC CTGGTGCCAGGTACTGTGTCAGGGCGGCCTCAGTG1AGGAGTGGCCAAAGGGTACTAGGGGTATGGGGAC CTGGTGCCAGGTACTGTGTCCGCCCGGCCTCAGTGAGCGAGCGAGCG!AGGGTACTAGGGGTATGGGGAC CTGGTGCCAGGTA1CTCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGTACTAGGGGTATGGGGAC Supplementry Figure 3 - Sequencing of trget site insertions nd deletions induced y ZFNs in vivo. PCR products from Cel-1 ssy on ZFN-treted mice in Fig.2c were cloned nd sequenced prior to tretment with Cel-1 enzyme. Sequencing of clones demonstrted, deletions nd, insertions chrcteristic of imprecise DSB repir y NHEJ. Red ses indicte ZFN inding sites while lue ses indicte the 6 p regionof ZFN clevge. Intervening white regions represent deleted ses while lck ses in etween ZFN inding sites indicte inserted ses. The 5 nd 3 flnking lck ses indicte hf9mut intron. The top line of ech pnel indictes the sequence of the unmodified locus. 3

4 RESEARCH SUPPLEMENTARY INFORMATION μg 2μg 4μg + ZFN Donor Trnsient trnsfection Single cell derived clones ZFN Donor 4μg - 2μg 4μg wt #1 #2 Undigested * Trgeted 3 k NotI clevge Untrgeted 2 k 6 8 % HDR k % HDR c % HDR gdna * in PCR input Trgeted Untrgeted % HDR quntified Supplementry Figure 4 - F9 ZFNs promote HDR t the hf9mut intron in HEK293T-hF9mut cells nd HDR cn e quntified through PCR. HEK293T cells were stly trnsduced with lentivirl vector crrying the hf9mut mini-gene nd single cell-derived clone ws expnded., Co-trnsfection of HEK293T-hF9mut cells with 400 ng of ZFN expression plsmid nd 2 μg or 4 μg of NotI RFLP donor plsmid results in gene trgeting 3 dys post-trnsfection, wheres trnsfection of 400 ng of ZFN expression plsmid lone, or 4 μg of donor plsmid does not result in detectle HDR., Trgeted integrtion of the splice cceptor exons 2-8 coding sequence ovine growth hormone polya signl cssette in HEK293T-hF9mut cells followed y the isoltion of either untrgeted ( wt ) or trgeted single cell-derived clones (#1 nd #2). (*) denotes non-specific mplicon present in ll smples. c, Linerity of the PCR-sed quntittive detection of HDR using primers P1/P3. Genomic DNA isolted from single cell-derived trgeted clone (#1) ws mixed t vrious rtios with wild-type (non-trgeted) gdna nd mplified y PCR using 32 P-leled nucleotides followed y PAGE nd utordiogrphy. The intensity of the nontrgeted nd trgeted nds were quntified. The % of detected trgeted signl is shown elow the gel nd the theoreticl signl is shown ove. 4

5 SUPPLEMENTARY INFORMATION RESEARCH Supplementry Figure 5 Week of life ZFN+Donor Mock+Donor ZFN Alone p-vlue hf9mut verge plsm hf.ix, ng/ml (n, std. dev.) (7, 117) 15 (6, 0) 15 (7, 0) (7, 111) 15 (6, 0) 15 (7, 0) (7, 122) 15 (6, 0) 15 (7, 0) wks 1 dy 156 (7, 140) 15 (6, 0) 15 (7, 0) wks 2 dys 205 (7, 170) 15 (6, 0) 15 (7, 0) (7, 149) 15 (6, 0) 15 (7, 0) (7, 165) 16 (6, 1) 15 (7, 0) (7, 123) 15 (6, 0) 15 (7, 0) (7, 116) 15 (6, 0) 15 (7, 0) (7, 144) 15 (6, 0) 15 (7, 0) (7, 136) 15 (6, 0) 15 (7, 0) (7, 133) 15 (6, 0) 15 (6, 0) (7, 223) 15 (6, 0) 17 (7, 4) 0.01 Week of life ZFN+Donor Mock+Donor p-vlue hf9mut/hb verge plsm hf.ix, ng/ml (n, std. dev.) (9, 96) 24 (9, 17) (8, 183) 21 (8, 10) (6, 160) 18 (4, 7) (5, 86) 16 (3, 2) (5, 38) 15 (3, 0) (5, 49) 18 (3, 6) Supplementry Figure 5 - Significnt difference in hf.ix expression etween groups., hf.ix expression in ZFN+Donor group t multiple time-points compred to ZFN lone nd Mock+Donor groups in hf9mut mice from Figure 4. p-vlues from 2-tiled T-test., hf.ix expression in ZFN+Donor group t multiple time-points compred to Mock+ Donor group in hf9mut/hb mice from Figure 5. p-vlues from 2-tiled T-test. 5

6 RESEARCH SUPPLEMENTARY INFORMATION Supplementry Figure 6 P1 ZFN Alone Mock+Donor ZFN+Donor P2 Untrgeted Trgeted P1 % HDR P3 Trgeted Untrgeted % HDR Clevge (Cel-I) % Indels Plsm hf.ix (ng/ml) R2 = Supplementry Figure 6 - Correltion of ZFN ctivity, HDR efficiency, nd hf.ix expression in hf9mut mice., Quntifiction of HDR efficiency using primers P1/P2 nd P1/P3 PCR ssys, quntifiction of ZFN-induced indel frequency using the Cel-I ssy, nd mesurement of plsm hf.ix levels y ELISA in mice from Fig.4 t 32 weeks of life demonstrtes correltion of ZFN clevge ctivity with HDR efficiency nd hf.ix expression. PCR ws performed using 32P-leled nucleotides, followed y PAGE nd product nd intensity quntifiction y utordiogrphy. Lnes with no quntifiction were elow the limit of detection. Ech lne represents n individul mouse., Liner regression of plsm hf.ix versus HDR trgeting rte, s mesured using primers P1/P2, demonstrtes ssocition etween degree of HDR nd level of hf.ix expression. 6 W W W. N A T U R E. C O M / N A T U R E

7 SUPPLEMENTARY INFORMATION RESEARCH hf9mut untrgeted hf9mut trgeted C57BL/6-wt ZFN lone Mock+donor ZFN+donor hf9mut trgeted hf9mut untrgeted C57BL/6-wt ZFN lone Mock+donor ZFN+donor P1 P1 Trgeted Untrgeted P3 Untrgeted Trgeted P2 c hf9mut trgeted hf9mut untrgeted C57BL/6-wt ZFN lone Mock+donor ZFN+donor mf8 control PCR Supplementry Figure 7 - Lck of mplifiction products for PCR-sed HDR quntifiction ssys in C57BL/6-wt mice. Liver genomic DNA from control mice in Fig.4c ws PCR mplified using, primers P1/P3,, primers P1/P2, or c, endogenous mouse F8 gene (to show no inhiition of PCR) primers. PCR ws performed using 32 P-leled nucleotides, followed y PAGE nd product nd detection y, utordiogrphy, or -c, ethidium romide stining. Ech lne represents n individul mouse. 7

8 RESEARCH SUPPLEMENTARY INFORMATION p=0.35 PHx p=0.09 p=0.12 p=0.01 c Week of life ZFN:Donor, 1:5 ZFN:Donor, 1:1 p-vlue hf9mut/hb verge plsm hf.ix, ng/ml (n, std. dev.) (9, 96) 100 (7, 70) (8, 183) 88 (7, 63) (6, 160) 119 (7, 101) (5, 86) 98 (6, 58) (5, 38) 105 (6, 60) (5, 49) 116 (6, 68) Supplementry Figure 8 - hf.ix expression nd PTT correction re dependent on Donor dose., Plsm hf.ix in hf9mut/hb mice following dy 2 of life I.P. injection with ZFN lone (5e10vg), Mock(5e10vg)+Donor(2.5e11vg), ZFN(5e10vg)+Donor(2.5e11vg), or ZFN (5e10vg)+Donor(5e10vg). Error rs denote std. error. PHx=prtil heptectomy., Test of clot formtion y PTT t week 14 of life for mice from pnel s well s WT nd HB control mice. p-vlues from 2-tiled T-test. c, hf.ix expression in ZFN:Donor, 1:5 group t multiple time-points compred to ZFN:Donor, 1:1 group in hf9mut/hb mice. p-vlues from 2-tiled T-test. 8

9 SUPPLEMENTARY INFORMATION RESEARCH Predicted!"#$%&'#$()*('+",#'(-%'# ZFN trget site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upplementry Figure 9 - Off-trget clevge y F9 ZFNs in vivo. Potentil off-trget clevge sites in the mouse genome s determined using SELEX pproch. Mismtches re indicted y lower-cse letters. To determine the frequency of off-trget ZFN-induced insertions nd deletions, the Cel-I ssy ws performed t ech site using liver genomic DNA of mice from Figure 2c nd verged mong the 4 smples. 9

10 RESEARCH SUPPLEMENTARY INFORMATION AAV-Donor: Homology SA Exons2-8 LM-PCR nested primers PolyA Homology Stuffer Integrtion within genes Integrtion within exons Integrtion ner oncogenes Mock+Donor ZFN+Donor ZFn+Donor 0.8 Mock+Donor ZFN+Donor ZFn+Donor 0.8 Mock+Donor ZFN+Donor ZFn+Donor proportion of sites proportion in exons proportion of sites In Gene Not in Gene Not In Exon In Exon <50k >50k Supplementry Figure 10- LM-PCR cloning nd 454 pyrosequencing to chrcterize AAV-F9 donor integrtion junctions t off-trget sites. We used the strtegy descried in Li et l., 2011 to chrcterize AAV-Donor integrtion sites in the mouse genome t sites distnt from the hf9 trget site., LM-PCR strtegy to clone NHEJ-medited events y using donor vector primers hyridizing to the stuffer. The stuffer is outside the rms of homology nd thus is eliminted y HDR events. This pproch thus mps integrtion sites where the stuffer remined intct (i.e. NHEJdependent integrtions) nd could not query HDR-medited events ecuse 454 reds would not spn the length of the rms of homology. To crete the second primer inding site, genomic DNA ws cleved with restriction enzyme nd ligted to linker, which ws then used s the inding site for the reverse PCR primer., Integrtion site preferences for AAV Donor integrtions within the mouse genome. One plte of 454/Roche Titnium sequencing ws used to query integrtion sites from five mice treted with ZFN + Donor, nd five control mice treted with Mock + Donor. The numer of junction sequence reds ligning to unique sites in the mouse genome in ech group were 5801 (ZFN + Donor) or (Mock + Donor). The reltive likelihood of donor integrtions to occur within genes, within exons, nd within 50k of oncogenes reltive to rndomly selected sites within the mouse genome 30 re indicted y the r grphs (red for Mock + Donor nd green for ZFN + Donor). Events re more likely to occur thn rndom if ove the lck line nd less likely if elow the lck line. No significnt differences were detected in the presence or sence of the AAV-ZFN vector for these comprisons, indicting tht ZFN clevge t off-trget sites did not hve strong effect on vector integrtion. Sequence dt re ville from the Ntionl Center for Biotechnology Informtion (NCBI) Genome Survey Sequences Dtse (GSSS, ccession numers: HR HR999999, nd JJ JJ725186). 10

11 SUPPLEMENTARY INFORMATION RESEARCH Untrgeted hf9mut ATG P1 Exon1 Liver Promoter ZFN inding site Intron Exons2-6 Y155stop Exons7-8 Donor Vector Homology SA Exons2-8 PolyA Homology Stuffer Integrted Donor vector ATG P1 Exon1 Liver Promoter Int- P4 Homology SA Exons2-8 Integrtion t DSB PolyA Homology Stuffer -ron Exons2-6 P4 Mock + Donor ZFN + Donor P1 P4 NHEJ-driven Integrtion Supplementry Figure 11 - Cpture of vector genome t the ZFN trget site., Vector DNA trgeted integrtion t the ZFN trget site cn e detected through PCR using primers P1 nd P4., PCR nlysis with primer pir P1/P4 showing vector integrtion t the ZFN trget site. PCR ws performed using 32 P-leled nucleotides, followed y PAGE nd utordiogrphed. 11

12 RESEARCH SUPPLEMENTARY INFORMATION p = 0.19 p = 0.29 p = 0.76 p = 0.04 c 1 week 3 weeks 8 weeks nti-flag (ZFN) nti-flag (ZFN), overexpose nti-actin Supplementry Figure 12 - Kinetics of liver function tests nd ZFN expression fter neontl injection. Mesurement of plsm lnine minotrnsferse (ALT) y chromogenic ssy s mrker for liver dmge t vrious time-points following dy 2 of life I.P. injection of 2.5e11 v.g. AAV-Donor nd 5e10 v.g. of AAV-ZFN or AAV-Mock in, hf9mut nd, C57BL/6-wt mice. c, Western lotting to detect ZFN expression in whole liver lyste t weeks 1, 3, nd 8 post- dy 2 of life I.P. injection of 5e10 v.g. of AAV-ZFN. Note tht the loss of ZFN expression y 3 weeks of life likely contriutes to the sence of toxicity. 12

13 SUPPLEMENTARY INFORMATION RESEARCH ATG ZFN inding site Y155stop Untrgeted hf9mut Exon1 Liver Promoter Intron Exons2-6 Exons7-8 Donor Vector Homology SA Exons2-8 PolyA Homology Stuffer *p = *p = Supplementry Figure 13 - Expression of hf.ix in mice receiving ZFN nd Donor lcking exon 1 in the left rm of homology., Schemtic showing the mp of donor vector, distinct from the donor vector in Fig.3 in tht it does not contin exon 1 sequence within the 5 rm of homology., Plsm hf.ix levels in hf9mut mice following I.P. injection t dy 2 of life with either 5e10 v.g. AAV-ZFN lone (n=7), 5e10 v.g. AAV-ZFN nd 2.5e11 v.g. of the new AAV-Donor (n=8), or 5e10 v.g. AAV-Mock nd 2.5e11 v.g. of the new AAV-Donor (n=10). Plsm hf.ix ssyed y ELISA. Error rs denote stndrd error. P-vlues from 2-tiled T-test. 13

14 RESEARCH SUPPLEMENTARY INFORMATION Supplementry Figure 14 ATTTAAATGGCCGGCCAGTAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCA ACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTC AGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTG CCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCA CTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGA GGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGC CAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACG CTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGG CAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCT CCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCA CTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGAC AGTGAATGATCCCCCTGATCTGCGGCCTCGACGGTATCGATAAGCTTGATATCGAATTCTAGTCGTCGA CCACTTTCACAATCTGCTAGCAAAGGTTATGCAGCGCGTGAACATGATCATGGCAGAATCACCAGGCC TCATCACCATCTGCCTTTTAGGATATCTACTCAGTGCTGAATGTACAGGTTTGTTTCCTTTTTTAAAATAC ATTGAGTATGCTTGCCTTTTAGATATAGAAATATCTGATGCTGTCTTCTTCACTAAATTTTGATTACATGAT TTGACAGCAATATTGAAGAGTCTAACAGCCAGCACGCAGGTTGGTAAGTACTGGTTCTTTGTTAGCTAG GTTTTCTTCTTCTTCATTTTTAAAACTAAATAGATCGACAATGCTTATGATGCATTTATGTTTAATAAACACT GTTCAGTTCATGATTTGGTCATGTAATTCCTGTTAGAAAACATTCATCTCCTTGGTTTAAAAAAATTAAAA GTGGGAAAACAAAGAAATAGCAGAATATAGTGAAAAAAAATAACCACATTATTTTTGTTTGGACTTACCA CTTTGAAATCAAAATGGGAAACAAAAGCACAAACAATGGCCTTATTTACACAAAAAGTCTGATTTTAAGA TATATGACATTTCAAGGTTTCAGAAGTATGTAATGAGGTGTGTCTCTAATTTTTTAAATTATATATCTTCAAT TTAAAGTTTTAGTTAAAACATAAAGATTAACCTTTCATTAGCAAGCTGTTAGTTATCACCAAAGCTTTTCAT GGATTAGGAAAAAATCATTTTGTCTCTATGTCAAACATCTTGGAGTTGATATTTGGGGAAACACAATACT CAGTTGAGTTCCCTAGGGGAGAAAAGCAAGCTTAAGAATTGACATAAAGAGTAGGAAGTTAGCTAATGC AACATATATCACTTTGTTTTTTCACAACTACAGTGACTTTATGTATTTCCCAGAGGAAGGCATACAGGGA AGAAATTATCCCATTTGGACAAACAGCATGTTCTCACAGGAAGCATTTATCACACTTACTTGTCAACTTT CTAGAATCAAATCTAGTAGCTGACAGTACCAGGATCAGGGGTGCCAACCCTAAGCACCCCCAGAAAGC TGACTGGCCCTGGTGCCAGGTACTGTGTCAGGGTACTAGGGGTATGGGGACAGGTTAGTCCACCTGA GTGGGAAGAAGCGGTGAAAGGAGTCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGAGCTGGTCAT CCTCATCCTGATAAACTGCAAAAGGCTGCGGCAGTTACGCTAGGGATAACAGGGTAATATAGGGTGGTT CCCACTCCAGACATGATGTCAGCTGTGAAATCGACGTCGCTGGACCATAATTAGGCTTCTGTTCTTCAG GAGACATTTGTTCAAAGTCATTTGGGCAACCATATTCTGAAAACAGCCCAGCCAGGGTGATGGATCACT TTGCAAAGATCCTCAATGAGCTATTTTCAAGTGATGACAAAGTGTGAAGTTAACCGCTCATTTGAGAAC TTTCTTTTTCATCCAAAGTAAATTCAAATATGATTAGAAATCTGACCTTTTATTACTGGAATTCTCTTGACT AAAAGTAAAATTGAATTTTAATTCCTAAATCTCCATGTGTATACAGTACTGTGGGAACATCACAGATTTTG GCTCCATGCCCTAAAGAGAAATTGGCTTTCAGATTATTTGGATTAAAAACAAAGACTTTCTTAAGAGATG TAAAATTTTCATGATGTTTTCTTTTTTGCTAAAACTAAAGAATTATTCTTTTACATTTCAGTTTTTCTTGATC ATGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTC AAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACGAGAAGTTTTTGAA AACACTGAAAGAACAACTGAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGT TTAAATGGCGGCAGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGGA AAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCAGTTTTGTAAAAATAGTG CTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGAAAACCAGAAGTCCTGTGAA CCAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGGC TGTTTTTCCTGATGTGGACTATAGGGCCGGCCATTTAAAT Supplementry Figure S14 - hf9mut mini-gene sequence. Nucleotide sequence for the hf9mut construct. Underlined regions indicte liver enhncer nd promoter (ses ), hf9 exon 1 (ses ), ZFN trget site (ses ), nd hf9 exons 2-6 (ses ). 14