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Both TALENs and CRISPR/Cas9 directly target the HBB IVS2-654 (C > T) mutation in β-thalassemia-derived iPSCs.

Xu P, Tong Y, Liu XZ, Wang TT, Cheng L, Wang BY, Lv X, Huang Y, Liu DP - Sci Rep (2015)

Bottom Line: We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor.In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs.This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China [2] Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

No MeSH data available.


Related in: MedlinePlus

TALEN- or CRISPR/Cas9-targeted iPSCs retain normal pluripotency.(a) Karyotyping analysis of the TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs. Neither TALEN- nor CRISPR/Cas9-mediated gene editing in β-thalassemia iPSCs caused gross chromosomal alterations. (b) Immunostaining of ESC markers (Oct4, Sox2 and SSEA-4) in TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; 400X. Green, antigen staining; Blue, Hoechst. (c) EB formation from the TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; 100X Bright field. (d) HE staining of teratomas containing tissues of all three germ layers derived from TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; Scale bars, 100 μm.
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f4: TALEN- or CRISPR/Cas9-targeted iPSCs retain normal pluripotency.(a) Karyotyping analysis of the TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs. Neither TALEN- nor CRISPR/Cas9-mediated gene editing in β-thalassemia iPSCs caused gross chromosomal alterations. (b) Immunostaining of ESC markers (Oct4, Sox2 and SSEA-4) in TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; 400X. Green, antigen staining; Blue, Hoechst. (c) EB formation from the TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; 100X Bright field. (d) HE staining of teratomas containing tissues of all three germ layers derived from TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; Scale bars, 100 μm.

Mentions: We selected TALENs- and CRISPR/Cas9-targeted iPSC clones for further characterization. All targeted clones displayed typical iPSC morphology (Supplementary Figure S3) and remained normal karyotypes (Fig. 4a). Immunofluorescence analysis revealed that both targeted clones retained uniform expression of typical pluripotency markers such as OCT4, SOX2, and SSEA-4 (Fig. 4b). Furthermore, TALENs- and CRISPR/Cas9-targeted clones both formed typical embryonic bodies (EB) in vitro (Fig. 4c). In addition, to test their pluripotency in vivo, the targeted iPS clones were transplanted into severe combined immunodeficiency (SCID) mice, and teratoma formation was observed at 8 weeks. Histological examination revealed that the tumor comprised cell types from all three germ layers in both the TALENs- and CRISPR/Cas9-targeted iPS clones (Fig. 4d). These results suggest that β-thalassemia patient-derived iPSCs retain pluripotency after gene targeting by either TALENs or CRISPR/Cas9.


Both TALENs and CRISPR/Cas9 directly target the HBB IVS2-654 (C > T) mutation in β-thalassemia-derived iPSCs.

Xu P, Tong Y, Liu XZ, Wang TT, Cheng L, Wang BY, Lv X, Huang Y, Liu DP - Sci Rep (2015)

TALEN- or CRISPR/Cas9-targeted iPSCs retain normal pluripotency.(a) Karyotyping analysis of the TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs. Neither TALEN- nor CRISPR/Cas9-mediated gene editing in β-thalassemia iPSCs caused gross chromosomal alterations. (b) Immunostaining of ESC markers (Oct4, Sox2 and SSEA-4) in TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; 400X. Green, antigen staining; Blue, Hoechst. (c) EB formation from the TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; 100X Bright field. (d) HE staining of teratomas containing tissues of all three germ layers derived from TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; Scale bars, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496796&req=5

f4: TALEN- or CRISPR/Cas9-targeted iPSCs retain normal pluripotency.(a) Karyotyping analysis of the TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs. Neither TALEN- nor CRISPR/Cas9-mediated gene editing in β-thalassemia iPSCs caused gross chromosomal alterations. (b) Immunostaining of ESC markers (Oct4, Sox2 and SSEA-4) in TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; 400X. Green, antigen staining; Blue, Hoechst. (c) EB formation from the TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; 100X Bright field. (d) HE staining of teratomas containing tissues of all three germ layers derived from TALEN- and CRISPR/Cas9-targeted β-thalassemia iPSCs; Scale bars, 100 μm.
Mentions: We selected TALENs- and CRISPR/Cas9-targeted iPSC clones for further characterization. All targeted clones displayed typical iPSC morphology (Supplementary Figure S3) and remained normal karyotypes (Fig. 4a). Immunofluorescence analysis revealed that both targeted clones retained uniform expression of typical pluripotency markers such as OCT4, SOX2, and SSEA-4 (Fig. 4b). Furthermore, TALENs- and CRISPR/Cas9-targeted clones both formed typical embryonic bodies (EB) in vitro (Fig. 4c). In addition, to test their pluripotency in vivo, the targeted iPS clones were transplanted into severe combined immunodeficiency (SCID) mice, and teratoma formation was observed at 8 weeks. Histological examination revealed that the tumor comprised cell types from all three germ layers in both the TALENs- and CRISPR/Cas9-targeted iPS clones (Fig. 4d). These results suggest that β-thalassemia patient-derived iPSCs retain pluripotency after gene targeting by either TALENs or CRISPR/Cas9.

Bottom Line: We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor.In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs.This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China [2] Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

No MeSH data available.


Related in: MedlinePlus