76a494e14ee8e6aa5aee774c35281ce9 scirep_a2020m4v10p6997.pdf 5ac80acb797c89395b14faa3a475fda06e3b014c scirep_a2020m4v10p6997.pdf 34efb9f8fc01d60f32ac04ce9e93774594466b40bbbf743f700e9033fb1eb69d scirep_a2020m4v10p6997.pdf Title: TALEN mediated gene editing in a mouse model of Fanconi anemia Subject: Scientific Reports, doi:10.1038/s41598-020-63971-z Keywords: Creator: Springer Producer: iText® 5.3.5 ©2000-2012 1T3XT BVBA (SPRINGER SBM; licensed version) CreationDate: Fri Apr 17 16:26:28 2020 CEST ModDate: Mon Apr 20 13:28:10 2020 CEST Tagged: no UserProperties: no Suspects: no Form: AcroForm JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 782.362 pts Page rot: 0 File size: 2382928 bytes Optimized: no PDF version: 1.4 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JCGZYB+Corbel-Bold CID TrueType Identity-H yes yes yes 51 0 QYJASN+ArialUnicodeMS CID TrueType Identity-H yes yes yes 754 0 FNXHUJ+Corbel-BoldItalic CID TrueType Identity-H yes yes yes 52 0 MLTFHX+Corbel-BoldItalic2 Type 1C WinAnsi yes yes yes 318 0 MinionPro-Regular Type 1C Custom yes no yes 57 0 EDVPMB+Corbel-Bold2 Type 1C WinAnsi yes yes yes 55 0 MinionPro-It Type 1C Custom yes no yes 54 0 WPDKIP+Corbel2 Type 1C WinAnsi yes yes yes 58 0 TBKMIH+Corbel CID TrueType Identity-H yes yes yes 60 0 MyriadPro-It Type 1C WinAnsi yes no yes 61 0 EuclidSymbol Type 1C Custom yes no yes 53 0 MinionPro-Bold Type 1C WinAnsi yes no yes 56 0 QZKJSN+STIXGeneral-Regular Type 1C Custom yes yes yes 59 0 WPDKIP+Corbel2 Type 1C WinAnsi yes yes yes 638 0 KQNZMZ+Calibri-BoldItalic Type 1C WinAnsi yes yes no 80 0 JRYTYB+Arial-BoldMT Type 1C WinAnsi yes yes no 81 0 INJNKD+Arial-BoldItalicMT Type 1C WinAnsi yes yes no 82 0 EHLPGL+Calibri-Bold Type 1C WinAnsi yes yes no 83 0 KQNZMZ+Calibri-BoldItalic Type 1C WinAnsi yes yes no 84 0 GKKBIH+Calibri Type 1C WinAnsi yes yes no 85 0 EHLPGL+Calibri-Bold Type 1C WinAnsi yes yes no 86 0 FGAVUJ+Calibri-Italic Type 1C WinAnsi yes yes no 87 0 ZXYLOV+ArialMT Type 1C WinAnsi yes yes no 216 0 KQNZMZ+Calibri-BoldItalic Type 1C WinAnsi yes yes no 217 0 EHLPGL+Calibri-Bold Type 1C WinAnsi yes yes no 218 0 EHLPGL+Calibri-Bold Type 1C WinAnsi yes yes no 219 0 KQNZMZ+Calibri-BoldItalic Type 1C WinAnsi yes yes no 220 0 INJNKD+Arial-BoldItalicMT Type 1C WinAnsi yes yes no 221 0 EHLPGL+Calibri-Bold Type 1C WinAnsi yes yes no 222 0 EHLPGL+Calibri-Bold Type 1C WinAnsi yes yes no 223 0 EHLPGL+Calibri-Bold Type 1C WinAnsi yes yes no 250 0 KQNZMZ+Calibri-BoldItalic Type 1C WinAnsi yes yes no 251 0 Calibri-Bold2 Type 1C Custom yes no no 252 0 GKKBIH+Calibri Type 1C WinAnsi yes yes no 253 0 OEJSQR+SymbolMT Type 1C WinAnsi yes yes no 254 0 OEJSQR+TimesNewRomanPSMT Type 1C WinAnsi yes yes yes 455 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-09-24 02:43:23 CEST RepresentationInformation: scirep_a2020m4v10p6997.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-09-23 19:49:37 CEST Size: 2382928 Format: PDF Version: 1.4 Status: Well-Formed, but not valid SignatureMatches: PDF-hul ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used with th:56' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used with t:57' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used with :58' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used with:59' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used wit:60' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used wi:61' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used w:62' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 2. Differential length of nuclease-induced indels in WT vs. FA-A MEFs. All single deletions and insertions at the on-target site were analyzed for their length in nuclease treated WT- and FA-A MEF cells. The percentage of the different de:64' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the SV40pA :65' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the SV40pA:66' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the SV40p:67' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the SV40:68' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the SV4:69' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the SV:70' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the S:71' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the :72' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used :75' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the:76' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 4. Gene-targeting in WT murine HSPCs. (a) Flow chart indicating the study design and the different analyses performed in gene-edited murine HSPCs. (b) Schematic showing the architecture of the murine Mbs85 locus, with the target sit:80' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 4. Gene-targeting in WT murine HSPCs. (a) Flow chart indicating the study design and the different analyses performed in gene-edited murine HSPCs. (b) Schematic showing the architecture of the murine Mbs85 locus, with the target si:82' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 4. Gene-targeting in WT murine HSPCs. (a) Flow chart indicating the study design and the different analyses performed in gene-edited murine HSPCs. (b) Schematic showing the architecture of the murine Mbs85 locus, with the target s:83' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 4. Gene-targeting in WT murine HSPCs. (a) Flow chart indicating the study design and the different analyses performed in gene-edited murine HSPCs. (b) Schematic showing the architecture of the murine Mbs85 locus, with the target :84' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 4. Gene-targeting in WT murine HSPCs. (a) Flow chart indicating the study design and the different analyses performed in gene-edited murine HSPCs. (b) Schematic showing the architecture of the murine Mbs85 locus, with the target:85' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 4. Gene-targeting in WT murine HSPCs. (a) Flow chart indicating the study design and the different analyses performed in gene-edited murine HSPCs. (b) Schematic showing the architecture of the murine Mbs85 locus, with the targe:86' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used:87' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing th:88' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀攀搀 㨀㠀㤩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㐸㐮〸㌠㘰㌮㘵㘠㐹㈮㐰㘠㔹ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸㄠ〠刾㸾㸊敮摯扪਱㔱‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀攀搀㨀㤀〩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㌹㌮㠸㘠㔸㌮㘵㘠㐰㈮㤲㈠㔷ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸ㈠〠刾㸾㸊敮摯扪਱㔲‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀攀㨀㤀ㄩ⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㈶ㄮ㜶ㄠ㔲㌮㘵㘠㈷〮㈱ㄠ㔱ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸ㌠〠刾㸾㸊敮摯扪਱㔳‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀㨀㤀㈩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㐰㘮㠴㠠㔱㌮㘵㘠㐱㘮〱㘠㔰ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸ㐠〠刾㸾㸊敮摯扪਱㔴‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd: 34. Lombardo, A. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods 8, 861–869, https://doi.org/10.1038/nmeth.1674 (2011). :93' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀攀搀㨀㤀〩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㌹㌮㠸㘠㔸㌮㘵㘠㐰㈮㤲㈠㔷ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸ㈠〠刾㸾㸊敮摯扪਱㔲‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀攀㨀㤀ㄩ⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㈶ㄮ㜶ㄠ㔲㌮㘵㘠㈷〮㈱ㄠ㔱ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸ㌠〠刾㸾㸊敮摯扪਱㔳‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀㨀㤀㈩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㐰㘮㠴㠠㔱㌮㘵㘠㐱㘮〱㘠㔰ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸ㐠〠刾㸾㸊敮摯扪਱㔴‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd: 34. Lombardo, A. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods 8, 861–869, https://doi.org/10.1038/nmeth.1674 (2011). :93' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀攀㨀㤀ㄩ⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㈶ㄮ㜶ㄠ㔲㌮㘵㘠㈷〮㈱ㄠ㔱ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸ㌠〠刾㸾㸊敮摯扪਱㔳‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀㨀㤀㈩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㐰㘮㠴㠠㔱㌮㘵㘠㐱㘮〱㘠㔰ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸ㐠〠刾㸾㸊敮摯扪਱㔴‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd: 34. Lombardo, A. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods 8, 861–869, https://doi.org/10.1038/nmeth.1674 (2011). :93' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀琀㨀㤀㈩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㐰㘮㠴㠠㔱㌮㘵㘠㐱㘮〱㘠㔰ㄮ㔱㕝⽈⽎⽁值㰯丠ㄸ㐠〠刾㸾㸊敮摯扪਱㔴‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd: 34. Lombardo, A. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods 8, 861–869, https://doi.org/10.1038/nmeth.1674 (2011). :93' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd: 25. Diez, B. et al. Therapeutic gene editing in CD34+ hematopoietic progenitors from Fanconi anemia patients. EMBO Mol Med, https://doi.org/10.15252/emmm.201707540 (2017). :109' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 1. Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor use:117' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing t:118' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing :119' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing:120' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 3. Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containin:121' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 4. Gene-targeting in WT murine HSPCs. (a) Flow chart indicating the study design and the different analyses performed in gene-edited murine HSPCs. (b) Schematic showing the architecture of the murine Mbs85 locus, with the targ:125' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀挀㨀㄀㈀㜩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㈲㘮㐶㈠㘱㐮㜶㌠㈴ㄮ㘲‶〲⸶㈲崯䠯丯䅐㰼⽎′㤰‰⁒㸾㸾੥湤潢樊㈷㌠〠潢樊㰼⽆‴⽔祰支䅮湯琯䑥獴⣾＀匀瀀爀椀渀最攀爀一愀琀甀爀攀开匀挀椀刀攀瀀开㘀㌀㤀㜀㄀⸀椀渀搀搀㫾￾￾￾￾＀￾＀㈀㔀⻾￾＀￾＀䐀椀攀稀Ⰰ 䈀⸀⃾＀攀琀 愀泾＀⸀ 吀栀攀爀愀瀀攀甀琀椀挀 最攀渀攀 攀搀椀琀椀渀最 椀渀 䌀䐀㌀㐀⬀ 栀攀洀愀琀漀瀀漀椀攀琀椀挀 瀀爀漀最攀渀椀琀漀爀猀 昀爀漀洀 䘀愀渀挀漀渀椀 愀渀攀洀椀愀 瀀愀琀椀攀渀琀猀⸀⃾＀䔀䴀䈀伀 䴀漀氀 䴀攀擾＀Ⰰ⃾￾＀栀琀琀瀀猀㨀⼀⼀搀漀椀⸀漀爀最⼀㄀　⸀㄀㔀㈀㔀㈀⼀攀洀洀洀⸀㈀　㄀㜀　㜀㔀㐀ヾ￾￾￾＀ ㈀　㄀㜀⻾＀㨀㄀㈀㠩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㐳〮㜵㈠㔹㐮㜶㌠㐳㘮㐱㔠㔸㈮㘲㉝⽈⽎⽁值㰯丠㈹ㄠ〠刾㸾㸊敮摯扪ਲ㜴‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀㨀㄀㈀㤩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㔲ㄮ㜱㠠㔳㔮㌵㜠㔲㤮㜶㌠㔲ㄮ㜶㝝⽈⽎⽁值㰯丠㈹㈠〠刾㸾㸊敮摯扪ਲ㜵‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd: 12. Gonzalez-Murillo, A. et al. Development of lentiviral vectors with optimized transcriptional activity for the gene therapy of patients with Fanconi anemia. Hum Gene Ther 21, 623–630, https://doi.org/10.1089/hum.2009.141 (2010).:130' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd: 25. Diez, B. et al. Therapeutic gene editing in CD34+ hematopoietic progenitors from Fanconi anemia patients. EMBO Mol Med, https://doi.org/10.15252/emmm.201707540 (2017). :128' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 5. Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48⁜琀栀漀甀爀猀 瀀漀猀琀ⴀ渀甀挀氀攀漀昀攀挀琀椀漀渀⸀ 唀㨀 甀渀琀爀愀渀猀昀攀㨀㄀㈀㤩⽂潲摥牛〠〠そ⽓畢瑹灥⽌楮欯䉓㰼⽗‰⽔祰支䉯牤敲⽓⽓㸾⽒散瑛㔲ㄮ㜱㠠㔳㔮㌵㜠㔲㤮㜶㌠㔲ㄮ㜶㝝⽈⽎⽁值㰯丠㈹㈠〠刾㸾㸊敮摯扪ਲ㜵‰扪਼㰯䘠㐯呹灥⽁湮潴⽄敳琨SpringerNature_SciRep_63971.indd: 12. Gonzalez-Murillo, A. et al. Development of lentiviral vectors with optimized transcriptional activity for the gene therapy of patients with Fanconi anemia. Hum Gene Ther 21, 623–630, https://doi.org/10.1089/hum.2009.141 (2010).:130' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd:Figure 4. Gene-targeting in WT murine HSPCs. (a) Flow chart indicating the study design and the different analyses performed in gene-edited murine HSPCs. (b) Schematic showing the architecture of the murine Mbs85 locus, with the tar:132' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd: 25. Diez, B. et al. Therapeutic gene editing in CD34+ hematopoietic progenitors from Fanconi anemia patients. EMBO Mol Med, https://doi.org/10.15252/emmm.201707540 (2017). :134' cannot be found Offset: 1771343 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_63971.indd: 26. Rio, P. et al. Targeted gene therapy and cell reprogramming in Fanconi anemia. EMBO Mol Med 6, 835–848, https://doi.org/10.15252/emmm.201303374 (2014). :146' cannot be found Offset: 1771343 MIMEtype: application/pdf PDFMetadata: Objects: 858 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseOutlines Language: EN Outlines: Item: Title: TALEN mediated gene editing in a mouse model of Fanconi anemia Children: Item: Title: Results Children: Item: Title: Targeted genome integration in FA-A mouse embryonic fibroblasts. Item: Title: Generation of disease-free and functional FA-A MEF clones corrected by gene targeting in the Mbs85 locus. Item: Title: Efficient HDR-mediated gene editing in the mouse Mbs85 locus of WT HPCs. Item: Title: Phenotypic correction of gene-edited FA-A HPCs. Item: Title: Discussion Item: Title: Methods Children: Item: Title: Cells and cell culture. Item: Title: Transcription activator-like effector nuclease (TALEN), donors and control plasmids. Item: Title: Lipofection and nucleofection. Item: Title: Cell sorting & flow cytometry. Item: Title: Clonogenic -colony forming cell (CFC)- assays of hematopoietic progenitors and colony forming units (CFUs). Item: Title: Cel I (Surveyor) analysis of TALENs pairs activity. Item: Title: On-target integration analyses. Item: Title: Fluorescence in situ hybridization (FISH). Item: Title: Western blot. Item: Title: Chromosomal instability assay. Item: Title: Off-target analysis. Item: Title: Statistical analyses. Item: Title: Acknowledgements Item: Title: Figure 1 Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. Item: Title: Figure 2 Differential length of nuclease-induced indels in WT vs. Item: Title: Figure 3 Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. Item: Title: Figure 4 Gene-targeting in WT murine HSPCs. Item: Title: Figure 5 Gene-targeting in FA-A murine HSPCs. 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CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: FNXHUJ+Corbel-BoldItalic Flags: Symbolic, Italic FontBBox: -494, -239, 1194, 952 FontFile2: true Font: BaseFont: JCGZYB+Corbel-Bold CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: JCGZYB+Corbel-Bold Flags: Symbolic FontBBox: -556, -239, 1155, 952 FontFile2: true Font: BaseFont: QYJASN+ArialUnicodeMS CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: QYJASN+ArialUnicodeMS Flags: Symbolic FontBBox: -1011, -330, 2260, 1078 FontFile2: true XMP: application/pdf doi:10.1038/s41598-020-63971-z Springer US Scientific Reports, doi:10.1038/s41598-020-63971-z TALEN mediated gene editing in a mouse model of Fanconi anemia 10.1038/s41598-020-63971-z http://dx.doi.org/10.1038/s41598-020-63971-z journal Scientific Reports © 2020, The Author(s) 2045-2322 10.1038/s41598-020-63971-z Springer 2020-04-20T13:28:10+02:00 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OriginalDocumentID URI http://www.aiim.org/pdfa/ns/id/ pdfaid PDF/A ID Schema internal Part of PDF/A standard part Integer internal Amendment of PDF/A standard amd Text internal Conformance level of PDF/A standard conformance Text http://prismstandard.org/namespaces/basic/2.0/ prism Prism external The aggregation type specifies the unit of aggregation for a content collection. Comment PRISM recommends that the PRISM Aggregation Type Controlled Vocabulary be used to provide values for this element. Note: PRISM recommends against the use of the #other value currently allowed in this controlled vocabulary. In lieu of using #other please reach out to the PRISM group at info@prismstandard.org to request addition of your term to the Aggregation Type Controlled Vocabulary. aggregationType Text external Copyright copyright Text external The Digital Object Identifier for the article. The DOI may also be used as the dc:identifier. If used as a dc:identifier, the URI form should be captured, and the bare identifier should also be captured using prism:doi. If an alternate unique identifier is used as the required dc:identifier, then the DOI should be specified as a bare identifier within prism:doi only. If the URL associated with a DOI is to be specified, then prism:url may be used in conjunction with prism:doi in order to provide the service endpoint (i.e. the URL). doi Text external ISSN for an electronic version of the issue in which the resource occurs. Permits publishers to include a second ISSN, identifying an electronic version of the issue in which the resource occurs (therefore e(lectronic)Issn. If used, prism:eIssn MUST contain the ISSN of the electronic version. See prism:issn. issn Text external Title of the magazine, or other publication, in which a resource was/will be published. 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