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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

TALENs mediate higher homologous recombination efficiency than CRISPR/Cas9 in HBB loci in β-thalassemia patient-derived iPSCs.(a) Strategy for gene correction of the IVS2-654 C > T mutation using a TALEN or CRISPR/Cas9 combined with the piggyBac donor vector. A TALEN or CRISPR/Cas9 was used to induce a double-strand break (DSB) near the point mutation. The targeting construct of the piggyBac transposon carrying the selectable marker and flanked by 1000 bp of wild-type genomic sequences was used as a donor vector. In the gene targeting, clones that were correctly integrated into the HBB loci were selected by puromycin and further identified using 5’ junction and 3’ junction PCR primers. (b) Summary of successful integration events in TALEN- and CRISPR/Cas9-mediated gene targeting. The expanded clones represent successfully passaged clones, and the targeted clones represent positive clones in both 5’ junction and 3’ junction PCR (Supplementary Figure S2). (c) PCR and Sanger sequencing analysis of site-specific homologous recombination mediated by TALENs in β-thalassemia iPSCs. Agorose gel results were cropped from the full-length gels which were presented in Supplementary Figure S5.B and all the gels were run under the same condition. Two pairs of PCR primers (each pair contained one primer corresponding to piggyBac and a second corresponding to HBB outside the targeting construct) were used to detect integration events. Sanger sequencing further confirmed correct integration. (d) PCR and Sanger sequencing for analyzing site-specific homologous recombination mediated by CRISPR/Cas9 in β-thalassemia iPSCs. Agorose gel results were cropped from the full-length gels which were presented in Supplementary Figure S5.C and all the gels were run under the same condition.
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f2: TALENs mediate higher homologous recombination efficiency than CRISPR/Cas9 in HBB loci in β-thalassemia patient-derived iPSCs.(a) Strategy for gene correction of the IVS2-654 C > T mutation using a TALEN or CRISPR/Cas9 combined with the piggyBac donor vector. A TALEN or CRISPR/Cas9 was used to induce a double-strand break (DSB) near the point mutation. The targeting construct of the piggyBac transposon carrying the selectable marker and flanked by 1000 bp of wild-type genomic sequences was used as a donor vector. In the gene targeting, clones that were correctly integrated into the HBB loci were selected by puromycin and further identified using 5’ junction and 3’ junction PCR primers. (b) Summary of successful integration events in TALEN- and CRISPR/Cas9-mediated gene targeting. The expanded clones represent successfully passaged clones, and the targeted clones represent positive clones in both 5’ junction and 3’ junction PCR (Supplementary Figure S2). (c) PCR and Sanger sequencing analysis of site-specific homologous recombination mediated by TALENs in β-thalassemia iPSCs. Agorose gel results were cropped from the full-length gels which were presented in Supplementary Figure S5.B and all the gels were run under the same condition. Two pairs of PCR primers (each pair contained one primer corresponding to piggyBac and a second corresponding to HBB outside the targeting construct) were used to detect integration events. Sanger sequencing further confirmed correct integration. (d) PCR and Sanger sequencing for analyzing site-specific homologous recombination mediated by CRISPR/Cas9 in β-thalassemia iPSCs. Agorose gel results were cropped from the full-length gels which were presented in Supplementary Figure S5.C and all the gels were run under the same condition.

Mentions: To correct the IVS2-654 C > T mutation in the HBB gene of β-thalassemia derived iPSCs, we constructed a targeting donor vector by inserting two nearly 1-kb segments upstream and downstream of the TTAA sequence at intron2 of HBB. The donor vector contains two ITRs of the piggyBac transposon and a bi-functional hybrid puro TK gene for positive and negative selection23. The β-thalassemia iPSCs were first transfected with either TALENs or CRISPR/Cas9 in the presence of donor plasmids. After selection with PUROMYCIN for approximately 12 days, the drug-resistant colonies were isolated, propagated and further tested for homologous recombination by junction PCR amplification using two pairs of primers to screen positive colonies (Fig. 2a).


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)

TALENs mediate higher homologous recombination efficiency than CRISPR/Cas9 in HBB loci in β-thalassemia patient-derived iPSCs.(a) Strategy for gene correction of the IVS2-654 C > T mutation using a TALEN or CRISPR/Cas9 combined with the piggyBac donor vector. A TALEN or CRISPR/Cas9 was used to induce a double-strand break (DSB) near the point mutation. The targeting construct of the piggyBac transposon carrying the selectable marker and flanked by 1000 bp of wild-type genomic sequences was used as a donor vector. In the gene targeting, clones that were correctly integrated into the HBB loci were selected by puromycin and further identified using 5’ junction and 3’ junction PCR primers. (b) Summary of successful integration events in TALEN- and CRISPR/Cas9-mediated gene targeting. The expanded clones represent successfully passaged clones, and the targeted clones represent positive clones in both 5’ junction and 3’ junction PCR (Supplementary Figure S2). (c) PCR and Sanger sequencing analysis of site-specific homologous recombination mediated by TALENs in β-thalassemia iPSCs. Agorose gel results were cropped from the full-length gels which were presented in Supplementary Figure S5.B and all the gels were run under the same condition. Two pairs of PCR primers (each pair contained one primer corresponding to piggyBac and a second corresponding to HBB outside the targeting construct) were used to detect integration events. Sanger sequencing further confirmed correct integration. (d) PCR and Sanger sequencing for analyzing site-specific homologous recombination mediated by CRISPR/Cas9 in β-thalassemia iPSCs. Agorose gel results were cropped from the full-length gels which were presented in Supplementary Figure S5.C 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

f2: TALENs mediate higher homologous recombination efficiency than CRISPR/Cas9 in HBB loci in β-thalassemia patient-derived iPSCs.(a) Strategy for gene correction of the IVS2-654 C > T mutation using a TALEN or CRISPR/Cas9 combined with the piggyBac donor vector. A TALEN or CRISPR/Cas9 was used to induce a double-strand break (DSB) near the point mutation. The targeting construct of the piggyBac transposon carrying the selectable marker and flanked by 1000 bp of wild-type genomic sequences was used as a donor vector. In the gene targeting, clones that were correctly integrated into the HBB loci were selected by puromycin and further identified using 5’ junction and 3’ junction PCR primers. (b) Summary of successful integration events in TALEN- and CRISPR/Cas9-mediated gene targeting. The expanded clones represent successfully passaged clones, and the targeted clones represent positive clones in both 5’ junction and 3’ junction PCR (Supplementary Figure S2). (c) PCR and Sanger sequencing analysis of site-specific homologous recombination mediated by TALENs in β-thalassemia iPSCs. Agorose gel results were cropped from the full-length gels which were presented in Supplementary Figure S5.B and all the gels were run under the same condition. Two pairs of PCR primers (each pair contained one primer corresponding to piggyBac and a second corresponding to HBB outside the targeting construct) were used to detect integration events. Sanger sequencing further confirmed correct integration. (d) PCR and Sanger sequencing for analyzing site-specific homologous recombination mediated by CRISPR/Cas9 in β-thalassemia iPSCs. Agorose gel results were cropped from the full-length gels which were presented in Supplementary Figure S5.C and all the gels were run under the same condition.
Mentions: To correct the IVS2-654 C > T mutation in the HBB gene of β-thalassemia derived iPSCs, we constructed a targeting donor vector by inserting two nearly 1-kb segments upstream and downstream of the TTAA sequence at intron2 of HBB. The donor vector contains two ITRs of the piggyBac transposon and a bi-functional hybrid puro TK gene for positive and negative selection23. The β-thalassemia iPSCs were first transfected with either TALENs or CRISPR/Cas9 in the presence of donor plasmids. After selection with PUROMYCIN for approximately 12 days, the drug-resistant colonies were isolated, propagated and further tested for homologous recombination by junction PCR amplification using two pairs of primers to screen positive colonies (Fig. 2a).

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