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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
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Genome Engineering for Stem Cell Transplantation.

Hassan Argani1

  • 1From the Shahidbeheshti University of Medical Sciences, Tehran, Iran.

Experimental and Clinical Transplantation : Official Journal of the Middle East Society for Organ Transplantation
|February 20, 2019
PubMed
Summary

Genome engineering with CRISPR-Cas9 offers ethical alternatives to embryonic stem cells. This technology enables precise gene editing in human pluripotent stem cells for research and potential therapies.

Area of Science:

  • Stem cell biology
  • Genome engineering
  • Epigenetics

Background:

  • Ethical concerns surrounding embryonic stem cells have driven research towards inducible pluripotent stem cells (iPSCs).
  • CRISPR-Cas9 technology provides a powerful and versatile tool for precise genome editing.
  • Understanding pluripotency markers and epigenetic regulation is crucial for stem cell research.

Purpose of the Study:

  • To explore the application of CRISPR-Cas9 genome engineering in human pluripotent stem cells.
  • To investigate the potential of combining CRISPR-Cas9 with iPSCs for gene manipulation and studying cellular processes.
  • To highlight the implications of RNA-guided genome targeting for synthetic biology and gene therapy.

Main Methods:

  • Utilized the CRISPR-Cas9 system for gene knockout and knock-in manipulations in human pluripotent stem cells.

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  • Employed homologous and nonhomologous DNA repair pathways for genome editing.
  • Applied nonviral delivery methods, including lipid-mediated transfection and electroporation, for efficient vector delivery.

    • Main Results:

    • Demonstrated the successful generation of single or multiple gene knockouts, mutation correction, and reporter transgene insertion.
    • Showcased the utility of gene knockouts for investigating epigenetic roles, such as DNA methylation.
    • Established CRISPR-Cas9 as a method to create modified Cas9 proteins for advanced applications.

    Conclusions:

    • CRISPR-Cas9 combined with iPSCs provides a robust platform for studying stem cell differentiation, lineage choice, and gene function.
    • This approach facilitates the investigation of genetic and noncoding elements influencing stem cell fate.
    • RNA-guided genome targeting holds significant promise for synthetic biology, gene network perturbation, and advanced gene therapies.