Engineering self-deliverable ribonucleoproteins for genome editing in the brain

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Editing Technologies

Background:

• CRISPR ribonucleoproteins (RNPs) offer advantages for genome editing but face delivery challenges like low efficiency and cell toxicity.
• Effective delivery of RNPs is crucial for advancing in vitro and in vivo genome editing applications.

Purpose of the Study:

• To engineer self-deliverable CRISPR-Cas9 RNPs that enhance cellular uptake and genome editing efficiency without auxiliary materials.
• To identify and optimize cell-penetrating peptides (CPPs) for improved RNP delivery and editing performance.

Main Methods:

• Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein to identify potent delivery constructs.
• Engineering of Cas9 fusion proteins, specifically a C-terminal fusion with three copies of the A22p peptide.
• Direct injection of self-deliverable Cas9 RNPs into the mouse striatum for in vivo validation.

Main Results:

• Identified potent CRISPR-Cas9-CPP fusions capable of efficient genome editing in neural progenitor cells.
• Developed an optimized construct (Cas9 fused to three A22p peptides) demonstrating significantly improved editing efficacy.
• Demonstrated robust genome editing in clinically relevant genes in vivo via direct striatal injection of self-deliverable Cas9 RNPs.

Conclusions:

• Self-deliverable Cas9 RNPs represent a facile and effective platform for genome editing.
• Engineered RNPs overcome previous delivery limitations, enabling efficient in vitro and in vivo gene editing.
• This approach holds significant potential for therapeutic applications requiring precise genome modification.