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Surapol Issaragrisil - One of the best experts on this subject based on the ideXlab platform.

  • one step genetic correction of hemoglobin e beta thalassemia patient derived ipscs by the crispr cas9 system
    Stem Cell Research & Therapy, 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak Upratya, Surapol Issaragrisil
    Abstract:

    Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a Single-Stranded DNA Oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.

  • One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    'Springer Science and Business Media LLC', 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a Single-Stranded DNA Oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. Results The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future

Eric B Kmiec - One of the best experts on this subject based on the ideXlab platform.

  • insertional mutagenesis by crispr cas9 ribonucleoprotein gene editing in cells targeted for point mutation repair directed by short single stranded DNA Oligonucleotides
    PLOS ONE, 2017
    Co-Authors: Natalia Riveratorres, Kelly Banas, Pawel Bialk, Kevin Bloh, Eric B Kmiec
    Abstract:

    CRISPR/Cas9 and Single-Stranded DNA Oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and Single-Stranded DNA Oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the Single-Stranded Oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short Single-Stranded DNA Oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex.

Malcolm F. White - One of the best experts on this subject based on the ideXlab platform.

  • CC1, a novel crenarchaeal DNA binding protein
    Journal of Bacteriology, 2006
    Co-Authors: Uli Schwarz-linek, Reinhard Hensel, Bettina Siebers, Catherine H. Botting, Malcolm F. White
    Abstract:

    The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a Single-Stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a Single-Stranded DNA Oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind Single-Stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

  • CC1, a Novel Crenarchaeal DNA Binding Protein �
    2006
    Co-Authors: Xiao Luo, Uli Schwarz-linek, Reinhard Hensel, Bettina Siebers, Catherine H. Botting, Malcolm F. White
    Abstract:

    The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a Single-Stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a Single-Stranded DNA Oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind Single-Stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein. All organisms use chromatin proteins, which allow reversible compaction of their genomes in a manner that allows DNA replication, gene expression, recombination, and repair. In eukary

Jeffrey A Gralnick - One of the best experts on this subject based on the ideXlab platform.

  • efficient and precise genome editing in shewanella with recombineering and crispr cas9 mediated counter selection
    ACS Synthetic Biology, 2019
    Co-Authors: Anna D Corts, Lynn C Thomason, Ryan T Gill, Jeffrey A Gralnick
    Abstract:

    Dissimilatory metal-reducing bacteria, particularly those from the genus Shewanella, are of importance for bioremediation of metal contaminated sites and sustainable energy production. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Here we report the development of a highly efficient system based on Single-Stranded DNA Oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counter-selection. Our system uses two plasmids: a sgRNA targeting vector and an editing vector, the latter harboring both Cas9 and the phage recombinase W3 Beta. Following the experimental analysis of Cas9 activity, we demonstrate the ability of this system to efficiently and precisely engineer different Shewanella strains with an average efficiency of >90% among total transformed cells, compared to ≃5% by recombineering alone, and regardless of the gene modified. We also show that different genetic changes can be introduced: mismatche...

Methichit Wattanapanitch - One of the best experts on this subject based on the ideXlab platform.

  • one step genetic correction of hemoglobin e beta thalassemia patient derived ipscs by the crispr cas9 system
    Stem Cell Research & Therapy, 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak Upratya, Surapol Issaragrisil
    Abstract:

    Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a Single-Stranded DNA Oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.

  • One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
    'Springer Science and Business Media LLC', 2018
    Co-Authors: Methichit Wattanapanitch, Nattaya Damkham, Ponthip Potirat, Kongtana Trakarnsanga, Montira Janan, Pakpoom Kheolamai, Nuttha Klincumhom, Yaowalak U-pratya, Surapol Issaragrisil
    Abstract:

    Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a Single-Stranded DNA Oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. Results The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future