Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells
Authors: Jaichandran Sivalingam, Dimitar Kenanov, Hao Han, Ajit Johnson Nirmal, Wai Har Ng, Sze Sing Lee, Jeyakumar Masilamani, Toan Thang Phan, Sebastian Maurer-Stroh, Oi Lian Kon
Journal: Molecular Therapy (2015)

Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious. Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious. The bedrock of hemophilia A treatment is factor VIII (FVIII) protein replacement to restore hemostatic capacity to a level sufficient to enable normal blood coagulation during activities of daily living. Regular prophylaxis with plasma-free recombinant FVIII products is the treatment of choice as it greatly reduces the frequency of acute bleeding episodes, chronic musculoskeletal disability, and improves health-related quality of life.1Manco-Johnson MJ Abshire TC Shapiro AD Riske B Hacker MR Kilcoyne R et al.Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia.N Engl J Med. 2007; 357: 535-544Crossref PubMed Scopus (1463) Google Scholar,2Collins P Faradji A Morfini M Enriquez MM Schwartz L Efficacy and safety of secondary prophylactic vs. on-demand sucrose-formulated recombinant factor VIII treatment in adults with severe hemophilia A: results from a 13-month crossover study.J Thromb Haemost. 2010; 8: 83-89Crossref PubMed Scopus (120) Google Scholar Of the current global population of about 140,000 people with hemophilia A, 75% receive little or no FVIII replacement.3World Federation of Hemophilia Report on the Annual Global Survey 2012, 2013 World Federation of Hemophilia Montreal, Canada.Google Scholar Even when FVIII products are affordable, regular prophylaxis is associated with frequent breakthrough bleeding,4Collins PW Blanchette VS Fischer K Björkman S Oh M Fritsch S rAHF-PFM Study Group et al.Break-through bleeding in relation to predicted factor VIII levels in patients receiving prophylactic treatment for severe hemophilia A.J Thromb Haemost. 2009; 7: 413-420Crossref PubMed Scopus (262) Google Scholar while the need for frequent intravenous access limits acceptance, especially among children for whom effective early intervention is especially important.5Santagostino E Mancuso ME Barriers to primary prophylaxis in haemophilic children: the issue of the venous access.Blood Transfus. 2008; 6:Suppl 2: s12-s16PubMed Google Scholar The high cost of FVIII replacement products for more than half the world's population of hemophilia A patients motivates attempts to develop alternative therapies. In vivo gene therapy using viral vectors is appealing for FVIII deficiency. Although it has not yet achieved the same success as gene therapy for hemophilia B,6Nathwani AC Reiss UM Tuddenham EG Rosales C Chowdary P McIntosh J et al.Long-term safety and efficacy of factor IX gene therapy in hemophilia B.N Engl J Med. 2014; 371: 1994-2004Crossref PubMed Scopus (879) Google Scholar improvements in FVIII transgene expression and packaging in AAV vectors appear promising, as are approaches to minimize immune responses to AAV vectors. An alternative strategy is nonviral delivery of a FVIII transgene into autologous cells ex vivo. Proof of concept was demonstrated in a clinical trial of autologous dermal fibroblasts transfected ex vivo with a plasmid that delivered a B domain-deleted FVIII transgene.7Roth DA Tawa Jr, NE O'Brien JM Treco DA Selden RF Factor VIII Transkaryotic Therapy Study Group Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A.N Engl J Med. 2001; 344: 1735-1742Crossref PubMed Scopus (313) Google Scholar Since then, several programmable nucleases with the potential to modify genomes with high precision have emerged and can be delivered by nonviral vectors. Among these, zinc finger nuclease (ZFN) technology is currently most advanced towards possible clinical applications. A phase-1 clinical trial of ZFN-mediated CCR5 inactivation in autologous T cells reported no adverse event attributable to ZFN.8Tebas P Stein D Tang WW Frank I Wang SQ Lee G et al.Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.N Engl J Med. 2014; 370: 901-910Crossref PubMed Scopus (1008) Google Scholar Nonetheless, there is heightened awareness of potential oncogenic complications because clinical trials of transgene integration mediated by gammaretroviral vectors were marred by treatment-induced leukemias and myelodysplasia.9Hacein-Bey-Abina S Garrigue A Wang GP Soulier J Lim A Morillon E et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.J Clin Invest. 2008; 118: 3132-3142Crossref PubMed Scopus (1395) Google Scholar,10Stein S Ott MG Schultze-Strasser S Jauch A Burwinkel B Kinner A et al.Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease.Nat Med. 2010; 16: 198-204Crossref PubMed Scopus (631) Google Scholar,11Avedillo Díez I Zychlinski D Coci EG Galla M Modlich U Dewey RA et al.Development of novel efficient SIN vectors with improved safety features for Wiskott–Aldrich syndrome stem cell based gene therapy.Mol Pharm. 2011; 8: 1525-1537Crossref PubMed Scopus (67) Google Scholar The biosafety of all genome-modifying techniques is therefore crucial for clinical approval. Off-target genome modifications in ZFN-treated cells have not been comprehensively evaluated. They have been identified by screening bioinformatically predicted off-target sites,12Hockemeyer D Soldner F Beard C Gao Q Mitalipova M DeKelver RC et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.Nat Biotechnol. 2009; 27: 851-857Crossref PubMed Scopus (834) Google Scholar,13Perez EE Wang J Miller JC Jouvenot Y Kim KA Liu O et al.Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases.Nat Biotechnol. 2008; 26: 808-816Crossref PubMed Scopus (829) Google Scholar in vitro cleavage of biased libraries14Pattanayak V Ramirez CL Joung JK Liu DR Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection.Nat Methods. 2011; 8: 765-770Crossref PubMed Scopus (352) Google Scholar or sequencing the integration sites of integrase-defective lentiviral vectors.15Gabriel R Lombardo A Arens A Miller JC Genovese P Kaeppel C et al.An unbiased genome-wide analysis of zinc-finger nuclease specificity.Nat Biotechnol. 2011; 29: 816-823Crossref PubMed Scopus (430) Google Scholar These studies reported frequencies of off-target events ranging from 1 to 6%. A machine-learning classifier16Sander JD Ramirez CL Linder SJ Pattanayak V Shoresh N Ku M et al.In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites.Nucleic Acids Res. 2013; 41: e181Crossref PubMed Scopus (48) Google Scholar has partially resolved the issue of largely nonoverlapping off-target sites generated by different methods but there remain nontrivial method-dependent discrepancies in off-target site identifications.14Pattanayak V Ramirez CL Joung JK Liu DR Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection.Nat Methods. 2011; 8: 765-770Crossref PubMed Scopus (352) Google Scholar,15Gabriel R Lombardo A Arens A Miller JC Genovese P Kaeppel C et al.An unbiased genome-wide analysis of zinc-finger nuclease specificity.Nat Biotechnol. 2011; 29: 816-823Crossref PubMed Scopus (430) Google Scholar No single technique for interrogating the genome suffices to reveal off-target modifications comprehensively; neither are there consensus standards for assessing biosafety ex vivo.17Hendel A Fine EJ Bao G Porteus MH Quantifying on- and off-target genome editing.Trends Biotechnol. 2015; 33: 132-140Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar,18Pattanayak V Guilinger JP Liu DR Determining the specificities of TALENs, Cas9, and other genome-editing enzymes.Methods Enzymol. 2014; 546: 47-78Crossref PubMed Scopus (52) Google Scholar,19Koo T Lee J Kim JS Measuring and reducing off-target activities of programmable nucleases including CRISPR-Cas9.Mol Cells. 2015; 38: 475-481Crossref PubMed Scopus (145) Google Scholar We have adopted a suite of techniques comprising genomic analysis of bioinformatically predicted off-target sites, whole-genome sequencing (WGS), integration junction and donor vector copy number analysis, and RNA-seq transcriptome profiling to holistically evaluate the biosafety of AAVS1 ZFN-mediated FVIII transgene integration in primary human cells. We assembled codon-optimized AAVS1 ZFN monomers expressed from a single plasmid for stoichiometric expression of each monomer in transfected cells. This study aimed to address potential genotoxicity using AAVS1 ZFN designed for accurate and efficient on-target cleavage activity. In brief, monomers were mutated to suppress homodimerization,20Miller JC Holmes MC Wang J Guschin DY Lee YL Rupniewski I et al.An improved zinc-finger nuclease architecture for highly specific genome editing.Nat Biotechnol. 2007; 25: 778-785Crossref PubMed Scopus (813) Google Scholar and additional mutations were introduced to restore nuclease activity which is greatly reduced in obligate heterodimeric FokI catalytic domains.21Guo J Gaj T Barbas CF Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases.J Mol Biol. 2010; 400: 96-107Crossref PubMed Scopus (158) Google Scholar,22Doyon Y Vo TD Mendel MC Greenberg SG Wang J Xia DF et al.Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures.Nat Methods. 2011; 8: 74-79Crossref PubMed Scopus (320) Google Scholar The fully modified ZFN, named Enhanced Sharkey, could be expected to have substantially lower off-target activity (18.9% homodimer versus 1.7% heterodimer),20Miller JC Holmes MC Wang J Guschin DY Lee YL Rupniewski I et al.An improved zinc-finger nuclease architecture for highly specific genome editing.Nat Biotechnol. 2007; 25: 778-785Crossref PubMed Scopus (813) Google Scholar while mutations introduced to restore nuclease activity neither decrease nor increase cleavage specificity of the intended targets.21Guo J Gaj T Barbas CF Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases.J Mol Biol. 2010; 400: 96-107Crossref PubMed Scopus (158) Google Scholar,22Doyon Y Vo TD Mendel MC Greenberg SG Wang J Xia DF et al.Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures.Nat Methods. 2011; 8: 74-79Crossref PubMed Scopus (320) Google Scholar Thus, Enhanced Sharkey AAVS1 ZFN is novel in incorporating several molecular features that ought to favor accurate and efficient integration. We show that Enhanced Sharkey AAVS1 ZFN integrated a FVIII transgene and induced durable FVIII secretion by primary human umbilical cord-lining epithelial cells (CLECs) with negligible off-target effects. We tested three AAVS1 ZFN constructs: obligate heterodimer20Miller JC Holmes MC Wang J Guschin DY Lee YL Rupniewski I et al.An improved zinc-finger nuclease architecture for highly specific genome editing.Nat Biotechnol. 2007; 25: 778-785Crossref PubMed Scopus (813) Google Scholar; Sharkey21Guo J Gaj T Barbas CF Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases.J Mol Biol. 2010; 400: 96-107Crossref PubMed Scopus (158) Google Scholar and Enhanced Sharkey22Doyon Y Vo TD Mendel MC Greenberg SG Wang J Xia DF et al.Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures.Nat Methods. 2011; 8: 74-79Crossref PubMed Scopus (320) Google Scholar to quantify integration of pZDonor (50 bp) in K562 cells (Supplementary Figure S1 shows all plasmid constructs used). The obligate heterodimer was mutated as reported (E490K; I538K and Q486E; I499L) to disfavor homodimerization of FokI catalytic domains such that ZFN mainly functions as heterodimers. Two amino acid substitutions (S418P and K441E) in each monomer of the obligate heterodimer were introduced to enhance cleavage activity in the Sharkey variant. Enhanced Sharkey was a further modification to the Sharkey variant (H537R substitution in E490K; I538K; S418P; K441E nuclease and N496D substitution in Q486E; I499L; S418P; K441E nuclease) which was reported to confer superior cleavage activity while suppressing homodimerization. We further compared each construct under conditions of mild hypothermia (30 °C) and at 37 °C. Transient hypothermia increases the efficiency of ZFN-induced changes probably by decreasing global mRNA and protein turnover and enhancing viability of transfected cells.23Doyon Y Choi VM Xia DF Vo TD Gregory PD Holmes MC Transient cold shock enhances zinc-finger nuclease-mediated gene disruption.Nat Methods. 2010; 7: 459-460Crossref PubMed Scopus (120) Google Scholar Both Sharkey and Enhanced Sharkey ZFNs at 30 °C induced highest efficiencies of donor DNA integration (44.5 and 47.9%, respectively) assessed by restriction fragment length polymorphism (RFLP) (Figure 1). Enhanced Sharkey ZFN was used at 30 °C in all subsequent experiments. Under conditions of efficient transfection (Figure 2a, left panel), integration of 50-bp (Figure 2a, right panel), 3.75-kb (Figure 2b), and 9.1-kb (Figure 2c) donor DNAs into the AAVS1 locus of K562 cells was demonstrated by RFLP and integration junction PCR. Four out of 40 single-cell K562 clones transfected with the 9.1-kb donor were positive on screening by integration junction PCR, yielding an estimated 10% integration efficiency for large donor DNAs. Sequencing of integration junction PCR amplicons confirmed precise integration of donor DNA into intron 1 of PPP1R12C (Figure 2b,c). Cells electroporated with donor DNA alone in the absence of ZFNs did not show evidence of transgene integration by integration junction PCR and RFLP (Figure 2a,c). The 9.1-kb donor DNA delivered a hybrid human-porcine B domain-deleted FVIII cDNA (Supplementary Figure S2).24Sivalingam J Phan TT Kon OL Intragenic integration in DLC1 sustains factor VIII expression in primary human cells without insertional oncogenicity.Gene Ther. 2014; 21: 402-412Crossref PubMed Scopus (3) Google ScholarFigure 2AAVS1 locus-specific integration of different size donor DNAs. (a) ZFN-dependent integration of donor DNA. K562 cells were coelectroporated with pEGFP (reporter for transfection efficiency) and pZDonor with or without AAVS1 ZFN mRNA. (Left): Brightfield and fluorescence images of transfected cells. Bar = 100 µm (Right): PCR spanning the integration junction (top) and RFLP assay (bottom) performed on genomic DNA from cells 4 days after treatment with pZdonor only; or 4 (Day 4) and 16 days (Day 16) after treatment with pZdonor and AAVS1 ZFN mRNA provided evidence of site-specific integration of 50-bp donor DNA. Control PCR amplified a 900-bp region of the AAVS1 locus. (b) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZdonor EGFP. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells coelectroporated with pZDonor EGFP and Enhanced Sharkey ZFN with or without G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. (c) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZDonor Hybrid FVIII. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells electroporated with pZDonor Hybrid FVIII only or coelectroporated with Enhanced Sharkey ZFN followed by G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. White vertical lines in the gel images demarcate lanes that were merged for clarity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) RT-PCR showed highest levels of ZFN expression 8–48 hours after electroporation with Enhanced Sharkey AAVS1 ZFN plasmids (Supplementary Figure S3a). ZFN protein expression was also higher in CLECs subjected to transient (1–3 days) mild hypothermia after transfection compared to CLECs which were never exposed to hypothermia (Supplementary Figure S3b). ZFN activity was assessed by the CEL-1 assay (reporting DNA break repair by nonhomologous end joining), integration junction PCR and RFLP (evidence of site-specific donor integration). CEL-1 assay results showed significantly higher ZFN activity when right and left Enhanced Sharkey AAVS1 ZFN monomers were delivered as a single construct (43 ± 1.9%) compared to ZFN monomers delivered as two constructs (35 ± 1.8%) (P = 0.045) (Supplementary Figure S4a). Thus, subsequent experiments were conducted using a single plasmid that delivered both Enhanced Sharkey AAVS1 ZFN monomers and transient hypothermia. Under these conditions, integration junction PCR and RFLP analysis showed no donor DNA integration in CLECs electroporated with pZDonor only (Supplementary Figure S4b). Varying doses of Enhanced Sharkey AAVS1 ZFN and donor DNA were tested to determine conditions that induced least cellular toxicity quantified by cell viability and phosphorylated histone H2AX. The overall results showed that coelectroporation of 5–10 µg Enhanced Sharkey AAVS1 ZFN with 10 µg donor DNA induced least cellular toxicity (Supplementary Figure S5). As the size of FVIII transgene donor DNA was relatively large (9 kb) and because accurate integration was paramount, we first assessed the accuracy of genome targeting in CLECs using gene trap donor vectors, AAVS1 SA-2A-puromycin-pA, and AAV-CAGGS-EGFP,12Hockemeyer D Soldner F Beard C Gao Q Mitalipova M DeKelver RC et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.Nat Biotechnol. 2009; 27: 851-857Crossref PubMed Scopus (834) Google Scholar which delivered smaller sized donor DNAs. PCR of puromycin-resistant CLECs demonstrated integration of 1.3- and 4.2-kb donors, respectively, using genome-specific primers which overlapped the intended integration site (Supplementary figures S6 and S7). Sequencing of PCR amplicons confirmed complete integration of the 1.3-kb donor. Most of the integrated 4.2-kb donor was sequenced (except for a 1-kb GC-rich region within the CAGGS promoter), and showed no insertions, deletions or rearrangements. PCR of integration junctions and donor DNA were negative in untreated CLECs. Using the same gene trap strategy, integration junction and long PCR of CLECs coelectroporated with the FVIII transgene donor DNA and Enhanced Sharkey AAVS1 ZFN showed integration of the complete donor in intron 1 of PPP1R12C (Figure 3a,b), confirmed by AAVS1 site-specific PCR and sequencing of the complete FVIII transgene (Figure 3b,c; Supplementary Table S1). Puromycin-resistance selected CLECs with the integrated transgene (puro-CLECs). This selected population secreted substantially higher FVIII levels (2,131 ± 17 mU/106 cells/24 hours) 37 days postelectroporation than unselected CLECs (952 ± 8 mU/106 cells/24 hours) 1 day postelectroporation (Figure 3d). Quantifying on-target versus off-target transgene integrations by digital droplet PCR of integration junction amplicons (on-target) versus vector-specific amplicons (on- and off-target integrations) showed no significant difference in copy number, relative to control 1 diploid locus, of total vector (0.55 ± 0.11 copies) compared with left junction (0.57 ± 0.07 copies; P = 0.85) and right junction (0.65 ± 0.05 copies; P = 0.35) amplicons, suggesting minimal off-target integrations (Figure 3e). RT-PCR showed twofold lower PPP1R12C expression in puro-CLECs compared to untreated CLECs, consistent with monoallelic transgene integration (Figure 3f). Targeted deep sequencing of the predicted ten most likely off-target sites (OT1–OT10)12Hockemeyer D Soldner F Beard C Gao Q Mitalipova M DeKelver RC et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.Nat Biotechnol. 2009; 27: 851-857Crossref PubMed Scopus (834) Google Scholar (Supplementary Table S2) showed 4- and 1-bp deletions at low frequency (1.36 and 1.37%, respectively) only in OT1, an 8q24.3 intergenic locus. No other indels in OT2–OT10 specific to puro-CLECs were detected (Table 1). Sensitivity of indel detection was determined by spiking wild-type AAVS1 amplicon with a synthetic amplicon having a 5-bp deletion at the AAVS1 target site at molar ratios of 1:10 to 1:1,000 (mutant:wild type). Deep sequencing detected the spiked deletion at all ratios with a highly linear relationship between spike-in and observed levels (R2 = 0.999), establishing a detection sensitivity of 0.1% (Supplementary Figure S8).Table 1Targeted deep sequencing of in silico predicted 10 most likely AAVS1 ZFN off-target sitesWt-CLECPuro-CLECSiteChromosome locusChromosomal positionIndelsTotal reads mapped% IndelsIndelsTotal reads mapped% IndelsOT1Chr 8 Intergenic141507040−−−4 bp del10,5001.361415070721 bp del11,7077.541 bp del8,3936.341415070722 bp del11,7070.27−−−141507045−−−1 bp del10,4341.371415070721 bp insertion11,7072.831 bp insertion8,3932.531415070722 bp insertion11,7070.13−−−OT2Chr 10 Intergenic−None8,21780None93,0490OT3Chr 4 Intron 1 of RGS12−None4,98100None67,0830OT4Chr 10 Intergenic−None104,1860None110,3200OT5Chr 9 Intergenic1385634051 bp del53,2780.16None94,4640OT6Chr 14 Intron 1 of BEGAIN11010331421 bp del40,5700.181 bp del45,7370.14OT7Chr 7 Intron 14 of GRB10−None108,0220None107,5220OT8Chr 16 Intron 2 of NPIPL1/ Exon 1 of LAT−None89,9610None130,6440OT9Chr19 Intron 7 of STK1112247241 bp del57,4230.13None97,6370OT10Chr 12 Intron 4 of FAIM2−None97,6020None86,6600Top-10 predicted off-target sites for AAVS1 ZFNs (OT1-OT10; ref. 12Hockemeyer D Soldner F Beard C Gao Q Mitalipova M DeKelver RC et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.Nat Biotechnol. 2009; 27: 851-857Crossref PubMed Scopus (834) Google Scholar) were evaluated by targeted deep sequencing of amplicons from genomic DNA from Wt- CLECs and Puro-CLECs. Chromosomal loci of OT1-OT10, the corresponding chromosomal positions, total reads mapped, types of indels experimentally detected and their corresponding percentages are summarized. Indels present only in Puro-CLECs are highlighted in italics. All genome coordinates refer to hg19. Open table in a new tab Top-10 predicted off-target sites for AAVS1 ZFNs (OT1-OT10; ref. 12Hockemeyer D Soldner F Beard C Gao Q Mitalipova M DeKelver RC et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.Nat Biotechnol. 2009; 27: 851-857Crossref PubMed Scopus (834) Google Scholar) were evaluated by targeted deep sequencing of amplicons from genomic DNA from Wt- CLECs and Puro-CLECs. Chromosomal loci of OT1-OT10, the corresponding chromosomal positions, total reads mapped, types of indels experimentally detected and their corresponding percentages are summarized. Indels present only in Puro-CLECs are highlighted in italics. All genome coordinates refer to hg19. VarScan analysis25Koboldt DC Zhang Q Larson DE Shen D McLellan MD Lin L et al.VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing.Genome Res. 2012; 22: 568-576Crossref PubMed Scopus (2941) Google Scholar of WGS data generated 2,736 high-confidence indels specific to puro-CLECs, but none were in silico predicted off-target sites.26Cradick TJ Ambrosini G Iseli C Bucher P McCaffrey AP ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites.BMC Bioinformatics. 2011; 12: 152Crossref PubMed Scopus (36) Google Scholar We considered only loci harboring multiple indels to be true off-target events.27Gupta A Meng X Zhu LJ Lawson ND Wolfe SA Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases.Nucleic Acids Res. 2011; 39: 381-392Crossref PubMed Scopus (96) Google Scholar Genomic coordinates of high-confidence indels revealed 196 loci with 2–5 indels each. All were in repetitive DNA regions. Three off-target loci had the highest combined number of indels and single nucleotide polymorphisms (SNPs) (4–6) and also higher proportions (33–50%) of mapped reads than on-target indels (19–46%). On-target indels were identified by aligning all reads to a 980-base sequence of the reference AAVS1 locus (all genome coordinates refer to hg19). The ZFN cleavage site was between positions 401 and 430 of this sequence. Four of 21 mapped reads (19%) showed a single A deletion at position 400 of this sequence. Twenty-three of 50 mapped reads (46%) showed either a single T deletion or a single C insertion at position 407. The putative off-target events mapped to intergenic regions in chr2_237939600 (indel 1); chr21_15213700 (indel 2); and chr8_1365500 (indel 3). All lacked potential AAVS1 ZFN-binding sites. The nearest protein-coding genes were COPS8 (54 kb from indel 1), C21orf15 (1.7 kb from indel 2), and DLGAP2 (84 kb from indel 3). Indel 1 was a false-positive indel because sequence data showed only two single-base substitutions (Figure 4a). Indel 3 comprised insertions of two different satellite DNAs. The larger insertion (1,489 bp) was a 15-fold expansion of satellite DNA while the smaller insertion (186 bp) was a twofold expansion of a different satellite (Figure 4b). (Indel 2 could not be sequenced continuously owing to highly repetitive sequence motifs.) Tandem repeats are intrinsically unstable because of replication slippage and unequal sister chromatid exchange during mitosis. Off-target cleavage by FokI possibly facilitated repeat expansion by strand invasion. Our data indicate that off-target events caused by FokI dimerization independent of ZFN binding are rare. WGS analysis suggested four chromosomal rearrangements, all having unbalanced genome copy number. Three were interchromosomal and one was an intrachromosomal structural variant (Figure 4c). Genomic PCR repeatedly failed to detect all putative abnormal chromosomal junctions, suggesting that the rearrangements were another false positive finding. However, as it was important to determine if ZFN treatment had induced structural changes in chromosomes, we employed a different validation method based on relative genome copy number analysis. As all breakpoint loci in the four structural variants (SV) were predicted to have unbalanced genome copy number, we used quantitative PCR to determine the copy number of each