Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and <i>piggyBac</i>

Xie, Fei; Yan, Lin; Chang, Judy C.; Beyer, Ashley I.; Wang, Jiaming; Muench, Marcus O.; Kan, Yuet Wai

doi:10.1101/gr.173427.114

Cited by 387 publications

(333 citation statements)

References 41 publications

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“…Recent studies have shown that personalized iPSCs can be derived from b-thalassemia patient fibroblasts via the induction of transcription factors, and the mutations can be corrected using a transcription activator-like effector nuclease or clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 nuclease (CRISPR/Cas9) system [21][22][23]. However, these primed-state iPSCs derived from b-thalassemia patient fibroblasts have shown extremely low levels of single cell cloning efficiencies, thus impairing the subsequent targeting efficiencies.…”

Section: Introductionmentioning

confidence: 99%

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Yang

Zhang

et al. 2015

Stem Cells Translational Medicine

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Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self-renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with b-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross-species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene-correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:8-19 SIGNIFICANCEIn the present study, transgene-free naïve induced pluripotent stem cells (iPSCs) directly converted from the fibroblasts of a patient with b-thalassemia in a defined culture system were generated. These naïve iPSCs, which show ground-state pluripotency, exhibited significantly improved singlecell cloning ability, recovery capacity, and gene-targeting efficiency compared with conventional primed iPSCs. These results provide an improved strategy for personalized treatment of genetic diseases such as b-thalassemia.

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Section: Introductionmentioning

confidence: 99%

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Yang

Zhang

et al. 2015

Stem Cells Translational Medicine

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“…Induced pluripotent stem cells (iPSCs) derived from patient somatic cells, which can selfrenew indefinitely without losing the ability to differentiate into all cell types (5-9) and hold great promise for regenerative medicine, represent an ideal cell population for in situ correction of disease-causing mutations (10,11). Generation of ␤-Thal patient iPSCs, correction of the mutations housed in those iPSCs, and subsequent differentiation into HSCs offer an opportunity for autologous transplantation for disease treatment (12)(13)(14)(15)(16)(17)(18). Recently, the development of gene editing to correct the HBB mutation in ␤-Thal iPSCs followed by differentiation of the corrected iPSCs into HSCs offered a new therapeutic option for those who do not have a bone marrow match to that of potential donors (12,16,17).…”

mentioning

confidence: 99%

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells

Niu

Song

et al. 2016

Journal of Biological Chemistry

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␤-Thalassemia (␤-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific ␤-Thalinduced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin ␤ (HBB) gene CD41/ 42(؊CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in ␤-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in ␤-Thal iPSCs.

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“…Systemet har vist seg effektivt i korreksjon av sykdomsfremkallende genvarianter i iPS-celler isolert og laget fra pasienter med Duchenne muskeldystrofi 210 og β-thalassemi 211 . Genmodifiserte iPSC har ennå ikke vaert prøvet ut i klinikken.…”

Section: Faktaboks 66 Prinsippet For Crispr/cas9-effektorunclassified

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2017

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Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac

Cited by 387 publications

References 41 publications

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells

Mor på en ny måte

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