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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

CRISPR/Cas9 displays higher potential off-target efficiency than TALENs.(a) T7E1 assay for ten off-target sites potentially recognized by CRISPR/Cas9 in 293T cells. The DNA fragment including the off-target sequence was amplified using appropriate primers. The PCR products were then subjected to the T7E1 assay according to standard protocols. (b) T7E1 assay for ten potential off-target sites of TALENs in 293T cells. (c) T7E1 assay for ten potential off-target sites of CRISPR/Cas9 in β-thalassemia iPSCs. (d) T7E1 assay for ten potential off-target sites of the TALENs in β-thalassemia iPSCs. These agorose gel results were respectively cropped from the full-length gels which were presented in Supplementary Figure S5.D~G and all the gels were run under the same condition.
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f3: CRISPR/Cas9 displays higher potential off-target efficiency than TALENs.(a) T7E1 assay for ten off-target sites potentially recognized by CRISPR/Cas9 in 293T cells. The DNA fragment including the off-target sequence was amplified using appropriate primers. The PCR products were then subjected to the T7E1 assay according to standard protocols. (b) T7E1 assay for ten potential off-target sites of TALENs in 293T cells. (c) T7E1 assay for ten potential off-target sites of CRISPR/Cas9 in β-thalassemia iPSCs. (d) T7E1 assay for ten potential off-target sites of the TALENs in β-thalassemia iPSCs. These agorose gel results were respectively cropped from the full-length gels which were presented in Supplementary Figure S5.D~G and all the gels were run under the same condition.

Mentions: The potential off-target (OT) events of both TALENs and CRISPR/Cas9 were first measured in 293T cells by the T7E1 assay. We PCR amplified ten potential off-target loci in 293T cells transfected by CRISPR/Cas9. At least six sites (Site-2, 3, 4, 6, 8, 9) had obvious indels (Fig. 3a). PCR amplification and T7E1 were also performed in 293T cells transfected with TALENs, which revealed that only two target sites (Site-8 and 9) had been targeted to some extent (Fig. 3b). These results indicate that CRISPR/Cas9 had more obvious off-target events than TALENs in 293T cells.


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)

CRISPR/Cas9 displays higher potential off-target efficiency than TALENs.(a) T7E1 assay for ten off-target sites potentially recognized by CRISPR/Cas9 in 293T cells. The DNA fragment including the off-target sequence was amplified using appropriate primers. The PCR products were then subjected to the T7E1 assay according to standard protocols. (b) T7E1 assay for ten potential off-target sites of TALENs in 293T cells. (c) T7E1 assay for ten potential off-target sites of CRISPR/Cas9 in β-thalassemia iPSCs. (d) T7E1 assay for ten potential off-target sites of the TALENs in β-thalassemia iPSCs. These agorose gel results were respectively cropped from the full-length gels which were presented in Supplementary Figure S5.D~G and all the gels were run under the same condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: CRISPR/Cas9 displays higher potential off-target efficiency than TALENs.(a) T7E1 assay for ten off-target sites potentially recognized by CRISPR/Cas9 in 293T cells. The DNA fragment including the off-target sequence was amplified using appropriate primers. The PCR products were then subjected to the T7E1 assay according to standard protocols. (b) T7E1 assay for ten potential off-target sites of TALENs in 293T cells. (c) T7E1 assay for ten potential off-target sites of CRISPR/Cas9 in β-thalassemia iPSCs. (d) T7E1 assay for ten potential off-target sites of the TALENs in β-thalassemia iPSCs. These agorose gel results were respectively cropped from the full-length gels which were presented in Supplementary Figure S5.D~G and all the gels were run under the same condition.
Mentions: The potential off-target (OT) events of both TALENs and CRISPR/Cas9 were first measured in 293T cells by the T7E1 assay. We PCR amplified ten potential off-target loci in 293T cells transfected by CRISPR/Cas9. At least six sites (Site-2, 3, 4, 6, 8, 9) had obvious indels (Fig. 3a). PCR amplification and T7E1 were also performed in 293T cells transfected with TALENs, which revealed that only two target sites (Site-8 and 9) had been targeted to some extent (Fig. 3b). These results indicate that CRISPR/Cas9 had more obvious off-target events than TALENs in 293T cells.

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