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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

Restoration of HBB transcription after gene correction.(a) Agorose gel results show the deletion of selection marker and all the gels were run under the same condition. Primers for the 5’ and 3’ junctions and primers for puro△TK were used to measure the excision of piggyBac. A lack of amplification by both primer pairs indicates no piggyBac reintegration at the targeting site in the β-thalassemia iPS clone. Sanger sequencing confirmed the restoration of normal intron2 after removal. The red arrow indicates the 654C > T mutation. (b) Flow cytometric analysis of β-thalassemia hiPS-TALEN-corrected cells, β-thalassemia iPSCs and H1-ESCs using the surface markers CD34 and CD31. (c) A conventional RT-PCR assay was performed to amplify HBB cDNA in erythroblasts derived from β-thalassemia hiPS-TALEN corrected cells, β-thalassemia hiPS cells and H1-ESCs. Erythroblasts from Core CD34+ cells were used as a positive control. E: erythroblasts. The agorose gel result was cropped from the full-length gels which was presented in Supplementary Figure S5.H. The normal transcription length is 332 bp, and the transcript length in thalassemia cells was 405 bp. (d) RT-PCR analysis of the level of HBB gene expression (relative to GAPDH). Erythroblasts from Cord CD34+ cells were used as a positive control. The values are expressed as the mean ± SD of triplicate samples from a representative experiment.
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f5: Restoration of HBB transcription after gene correction.(a) Agorose gel results show the deletion of selection marker and all the gels were run under the same condition. Primers for the 5’ and 3’ junctions and primers for puro△TK were used to measure the excision of piggyBac. A lack of amplification by both primer pairs indicates no piggyBac reintegration at the targeting site in the β-thalassemia iPS clone. Sanger sequencing confirmed the restoration of normal intron2 after removal. The red arrow indicates the 654C > T mutation. (b) Flow cytometric analysis of β-thalassemia hiPS-TALEN-corrected cells, β-thalassemia iPSCs and H1-ESCs using the surface markers CD34 and CD31. (c) A conventional RT-PCR assay was performed to amplify HBB cDNA in erythroblasts derived from β-thalassemia hiPS-TALEN corrected cells, β-thalassemia hiPS cells and H1-ESCs. Erythroblasts from Core CD34+ cells were used as a positive control. E: erythroblasts. The agorose gel result was cropped from the full-length gels which was presented in Supplementary Figure S5.H. The normal transcription length is 332 bp, and the transcript length in thalassemia cells was 405 bp. (d) RT-PCR analysis of the level of HBB gene expression (relative to GAPDH). Erythroblasts from Cord CD34+ cells were used as a positive control. The values are expressed as the mean ± SD of triplicate samples from a representative experiment.

Mentions: To remove the drug-selectable cassette from a TALENs-targeted hiPSC clone, the cells were transiently re-expressed the piggyBac transposase. After negative selection by 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU), resistant colonies were picked and expanded. Genotyping was performed to detect the deletion of piggyBac transposon from the HBB locus (Fig. 5a, left panel). Sanger sequencing confirmed the seamless removal of the piggyBac transposon based on the restoration of the original intron2 with no exogenous sequence (Fig. 5a, right panel). These iPSC clones were morphologically normal and expressed pluripotency markers correctly(Data not shown).


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)

Restoration of HBB transcription after gene correction.(a) Agorose gel results show the deletion of selection marker and all the gels were run under the same condition. Primers for the 5’ and 3’ junctions and primers for puro△TK were used to measure the excision of piggyBac. A lack of amplification by both primer pairs indicates no piggyBac reintegration at the targeting site in the β-thalassemia iPS clone. Sanger sequencing confirmed the restoration of normal intron2 after removal. The red arrow indicates the 654C > T mutation. (b) Flow cytometric analysis of β-thalassemia hiPS-TALEN-corrected cells, β-thalassemia iPSCs and H1-ESCs using the surface markers CD34 and CD31. (c) A conventional RT-PCR assay was performed to amplify HBB cDNA in erythroblasts derived from β-thalassemia hiPS-TALEN corrected cells, β-thalassemia hiPS cells and H1-ESCs. Erythroblasts from Core CD34+ cells were used as a positive control. E: erythroblasts. The agorose gel result was cropped from the full-length gels which was presented in Supplementary Figure S5.H. The normal transcription length is 332 bp, and the transcript length in thalassemia cells was 405 bp. (d) RT-PCR analysis of the level of HBB gene expression (relative to GAPDH). Erythroblasts from Cord CD34+ cells were used as a positive control. The values are expressed as the mean ± SD of triplicate samples from a representative experiment.
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Related In: Results  -  Collection

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f5: Restoration of HBB transcription after gene correction.(a) Agorose gel results show the deletion of selection marker and all the gels were run under the same condition. Primers for the 5’ and 3’ junctions and primers for puro△TK were used to measure the excision of piggyBac. A lack of amplification by both primer pairs indicates no piggyBac reintegration at the targeting site in the β-thalassemia iPS clone. Sanger sequencing confirmed the restoration of normal intron2 after removal. The red arrow indicates the 654C > T mutation. (b) Flow cytometric analysis of β-thalassemia hiPS-TALEN-corrected cells, β-thalassemia iPSCs and H1-ESCs using the surface markers CD34 and CD31. (c) A conventional RT-PCR assay was performed to amplify HBB cDNA in erythroblasts derived from β-thalassemia hiPS-TALEN corrected cells, β-thalassemia hiPS cells and H1-ESCs. Erythroblasts from Core CD34+ cells were used as a positive control. E: erythroblasts. The agorose gel result was cropped from the full-length gels which was presented in Supplementary Figure S5.H. The normal transcription length is 332 bp, and the transcript length in thalassemia cells was 405 bp. (d) RT-PCR analysis of the level of HBB gene expression (relative to GAPDH). Erythroblasts from Cord CD34+ cells were used as a positive control. The values are expressed as the mean ± SD of triplicate samples from a representative experiment.
Mentions: To remove the drug-selectable cassette from a TALENs-targeted hiPSC clone, the cells were transiently re-expressed the piggyBac transposase. After negative selection by 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU), resistant colonies were picked and expanded. Genotyping was performed to detect the deletion of piggyBac transposon from the HBB locus (Fig. 5a, left panel). Sanger sequencing confirmed the seamless removal of the piggyBac transposon based on the restoration of the original intron2 with no exogenous sequence (Fig. 5a, right panel). These iPSC clones were morphologically normal and expressed pluripotency markers correctly(Data not shown).

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