Improving transgene expression and CRISPR-Cas9 efficiency with molecular engineering-based molecules.

Area of Science:

• Molecular Biology
• Immunology
• Gene Therapy

Background:

• The CRISPR-Cas9 system is a powerful gene-editing tool, often delivered via plasmid vectors.
• Foreign DNA from plasmids triggers an innate immune response (IIR), inhibiting transgene expression and gene editing.
• Existing methods to mitigate IIR are limited to single targets and can affect cell viability.

Purpose of the Study:

• To develop a comprehensive genetic approach to manipulate IIR for improved gene editing.
• To design and construct artificial nucleic acid molecules (ANAMs) targeting key IIR players.
• To evaluate the efficacy of ANAMs in enhancing transgene expression and CRISPR-Cas9 gene editing.

Main Methods:

• Designed and synthesized ANAMs, incorporating aptamers that bind to β-catenin and NF-κB, key components of IIR.
• Introduced ANAMs into mammalian cells to assess their impact on IIR.
• Utilized ANAMs in conjunction with the CRISPR-Cas9 system to evaluate gene editing efficiency and cancer cell apoptosis.

Main Results:

• ANAMs significantly inhibited IIR in cells, leading to enhanced transgene expression.
• The use of ANAMs improved the gene-editing efficiency of CRISPR-Cas9 and its derivatives.
• ANAMs enhanced CRISPR-Cas9-induced apoptosis in cancer cells.

Conclusions:

• ANAMs represent a novel genetic strategy for comprehensively modulating IIR.
• These molecules show promise for improving transgene expression and gene editing applications in mammalian systems.
• ANAMs can enhance the therapeutic potential of CRISPR-Cas9 gene editing, particularly in cancer therapy.