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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

Both TALENs and CRISPR/Cas9 can directly and efficiently target the HBB gene IVS2-654 mutation site.(a) Two pairs of TALENs and two sites of CRSIPR guide RNA were designed for in situ targeting of the HBB gene IVS2-654 mutation site. TALEN sites are indicated by blue lines, and guide RNA sites are indicated by red lines. The red nucleotide in the middle sequence is the IVS2-654 mutation site. PAM: protospacer adjacent motif. (b) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by a SSA (single strand annealing) assay. HEK293 cells were separately co-transfected with one of the pairs of TALENs or one of the pairs of CRISPRs and pSSA-HBB-IVS2 and TK-Renilla. At 48 h after transfection, the ratio of firefly luciferase and Renilla luciferase activity was measured by a microplate reader. TALEN-blank vector and pX330 blank vector were used as negative controls. The data represent the mean ± SD of three independent experiments. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (c) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 293T cells. The endogenous locus was amplified by PCR, and the product was further purified according to the manufacturer’s instructions. The purified PCR product was denatured and reannealed, and the hybridized PCR products were further digested by T7 Endonuclease I. The upper lane shows the separation of the DNA on a 2% agarose gel, this results were cropped from the full-length gels which were presented in Supplementary Figure S5.A and all the gels were run under the same condition; while the lower lane shows relative indel rates in different groups as measured with the ImageJ program. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (d) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 654hiPS cells. The upper lane shows the separation of DNA on a 2% agarose gel after T7E1 digestion; the lower lane shows the relative indel rates in different groups as measured by ImageJ. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (e) Sanger sequencing of the different mutant types of HBB intron2 in 293T cells after transfection with either TALEN or CRISPR/Cas9. The blue nucleotides in the upper lane represent two pairs of TALEN recognition sites. The red nucleotides in the lower lane represent the PAM (protospacer adjacent motif) sequence recognized by CRISPR/Cas9.
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f1: Both TALENs and CRISPR/Cas9 can directly and efficiently target the HBB gene IVS2-654 mutation site.(a) Two pairs of TALENs and two sites of CRSIPR guide RNA were designed for in situ targeting of the HBB gene IVS2-654 mutation site. TALEN sites are indicated by blue lines, and guide RNA sites are indicated by red lines. The red nucleotide in the middle sequence is the IVS2-654 mutation site. PAM: protospacer adjacent motif. (b) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by a SSA (single strand annealing) assay. HEK293 cells were separately co-transfected with one of the pairs of TALENs or one of the pairs of CRISPRs and pSSA-HBB-IVS2 and TK-Renilla. At 48 h after transfection, the ratio of firefly luciferase and Renilla luciferase activity was measured by a microplate reader. TALEN-blank vector and pX330 blank vector were used as negative controls. The data represent the mean ± SD of three independent experiments. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (c) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 293T cells. The endogenous locus was amplified by PCR, and the product was further purified according to the manufacturer’s instructions. The purified PCR product was denatured and reannealed, and the hybridized PCR products were further digested by T7 Endonuclease I. The upper lane shows the separation of the DNA on a 2% agarose gel, this results were cropped from the full-length gels which were presented in Supplementary Figure S5.A and all the gels were run under the same condition; while the lower lane shows relative indel rates in different groups as measured with the ImageJ program. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (d) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 654hiPS cells. The upper lane shows the separation of DNA on a 2% agarose gel after T7E1 digestion; the lower lane shows the relative indel rates in different groups as measured by ImageJ. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (e) Sanger sequencing of the different mutant types of HBB intron2 in 293T cells after transfection with either TALEN or CRISPR/Cas9. The blue nucleotides in the upper lane represent two pairs of TALEN recognition sites. The red nucleotides in the lower lane represent the PAM (protospacer adjacent motif) sequence recognized by CRISPR/Cas9.

Mentions: To induce DSBs near HBB loci, we designed two pairs of TALENs and two single--guide RNAs(sgRNA) to directly target the IVS2-654 C > T mutation (Fig. 1a).


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)

Both TALENs and CRISPR/Cas9 can directly and efficiently target the HBB gene IVS2-654 mutation site.(a) Two pairs of TALENs and two sites of CRSIPR guide RNA were designed for in situ targeting of the HBB gene IVS2-654 mutation site. TALEN sites are indicated by blue lines, and guide RNA sites are indicated by red lines. The red nucleotide in the middle sequence is the IVS2-654 mutation site. PAM: protospacer adjacent motif. (b) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by a SSA (single strand annealing) assay. HEK293 cells were separately co-transfected with one of the pairs of TALENs or one of the pairs of CRISPRs and pSSA-HBB-IVS2 and TK-Renilla. At 48 h after transfection, the ratio of firefly luciferase and Renilla luciferase activity was measured by a microplate reader. TALEN-blank vector and pX330 blank vector were used as negative controls. The data represent the mean ± SD of three independent experiments. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (c) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 293T cells. The endogenous locus was amplified by PCR, and the product was further purified according to the manufacturer’s instructions. The purified PCR product was denatured and reannealed, and the hybridized PCR products were further digested by T7 Endonuclease I. The upper lane shows the separation of the DNA on a 2% agarose gel, this results were cropped from the full-length gels which were presented in Supplementary Figure S5.A and all the gels were run under the same condition; while the lower lane shows relative indel rates in different groups as measured with the ImageJ program. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (d) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 654hiPS cells. The upper lane shows the separation of DNA on a 2% agarose gel after T7E1 digestion; the lower lane shows the relative indel rates in different groups as measured by ImageJ. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (e) Sanger sequencing of the different mutant types of HBB intron2 in 293T cells after transfection with either TALEN or CRISPR/Cas9. The blue nucleotides in the upper lane represent two pairs of TALEN recognition sites. The red nucleotides in the lower lane represent the PAM (protospacer adjacent motif) sequence recognized by CRISPR/Cas9.
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Related In: Results  -  Collection

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f1: Both TALENs and CRISPR/Cas9 can directly and efficiently target the HBB gene IVS2-654 mutation site.(a) Two pairs of TALENs and two sites of CRSIPR guide RNA were designed for in situ targeting of the HBB gene IVS2-654 mutation site. TALEN sites are indicated by blue lines, and guide RNA sites are indicated by red lines. The red nucleotide in the middle sequence is the IVS2-654 mutation site. PAM: protospacer adjacent motif. (b) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by a SSA (single strand annealing) assay. HEK293 cells were separately co-transfected with one of the pairs of TALENs or one of the pairs of CRISPRs and pSSA-HBB-IVS2 and TK-Renilla. At 48 h after transfection, the ratio of firefly luciferase and Renilla luciferase activity was measured by a microplate reader. TALEN-blank vector and pX330 blank vector were used as negative controls. The data represent the mean ± SD of three independent experiments. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (c) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 293T cells. The endogenous locus was amplified by PCR, and the product was further purified according to the manufacturer’s instructions. The purified PCR product was denatured and reannealed, and the hybridized PCR products were further digested by T7 Endonuclease I. The upper lane shows the separation of the DNA on a 2% agarose gel, this results were cropped from the full-length gels which were presented in Supplementary Figure S5.A and all the gels were run under the same condition; while the lower lane shows relative indel rates in different groups as measured with the ImageJ program. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (d) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 654hiPS cells. The upper lane shows the separation of DNA on a 2% agarose gel after T7E1 digestion; the lower lane shows the relative indel rates in different groups as measured by ImageJ. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) (e) Sanger sequencing of the different mutant types of HBB intron2 in 293T cells after transfection with either TALEN or CRISPR/Cas9. The blue nucleotides in the upper lane represent two pairs of TALEN recognition sites. The red nucleotides in the lower lane represent the PAM (protospacer adjacent motif) sequence recognized by CRISPR/Cas9.
Mentions: To induce DSBs near HBB loci, we designed two pairs of TALENs and two single--guide RNAs(sgRNA) to directly target the IVS2-654 C > T mutation (Fig. 1a).

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