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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
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CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression.

Mark J Osborn1,2,3,4,5, Christopher J Lees6, Amber N McElroy7

  • 1Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA. osbor026@umn.edu.

International Journal of Molecular Sciences
|March 23, 2018
PubMed
Summary
This summary is machine-generated.

This study demonstrates a novel CRISPR/Cas9 gene editing method to activate the COL7A1 gene in stem cells and T-cells. This approach offers a promising platform for gene therapy and cellular engineering.

Keywords:
CRISPR/Cas9T-cellsadeno-associated viruscord bloodhomology directed repairrecessive dystrophic epidermolysis bullosatranscriptional activationubiquitous chromatin opening element

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Area of Science:

  • Gene Therapy
  • CRISPR/Cas9 Gene Editing
  • Cellular Engineering

Background:

  • Gene and cellular therapies show promise for genetic disorders but face challenges in delivering large genes and achieving therapeutic expression.
  • Effective delivery and sustained expression of therapeutic genes in clinically relevant cells remain significant hurdles.

Purpose of the Study:

  • To engineer a CRISPR/Cas9 system for inserting regulatory elements to activate endogenous genes, specifically targeting the COL7A1 gene.
  • To develop a facile cellular engineering platform for promoting gene expression with high, sustained levels.

Main Methods:

  • Utilized the CRISPR/Cas9 system to insert regulatory elements (chromatin opening element, promoter, suicide gene) upstream of the COL7A1 gene.
  • Engineered primary human umbilical cord blood CD34⁺ hematopoietic stem cells and peripheral blood T-cells.
  • Assessed gene activation, stem cell functionality, and T-cell receptor complex disruption.

Main Results:

  • Achieved robust activation of the COL7A1 gene in both CD34⁺ cells and T-cells.
  • CD34⁺ cells maintained their colony-forming potential post-engineering.
  • Successfully disrupted the T-cell receptor complex in T-cells, enhancing their translational potential.

Conclusions:

  • Demonstrated the feasibility of targeted knock-in for precision gene activation using a CRISPR/Cas9-based system.
  • The developed system merges gene editing specificity with sustained gene expression, applicable to various genes and model systems.
  • This approach represents a versatile cellular engineering platform for advancing gene therapy strategies.