Modulating binding affinity of aptamer-based loading constructs enhances extracellular vesicle-mediated CRISPR/Cas9...

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Editing

Background:

• CRISPR/Cas9 gene editing requires efficient delivery of large Cas9-sgRNA ribonucleoprotein (RNP) complexes.
• Challenges in RNP delivery include immunogenicity, size, and negative charge.
• Extracellular vesicles (EVs) offer a natural platform for intracellular delivery of biomolecules.

Purpose of the Study:

• To investigate the use of EVs for delivering CRISPR/Cas9 Cas9-sgRNA RNP complexes.
• To explore the impact of modulating binding affinity between MS2 aptamers and MS2 coat proteins (MCPs) on RNP delivery efficiency.
• To determine if optimizing binding affinity can enhance functional RNP delivery without additional release strategies.

Main Methods:

• Engineered EVs to co-express Cas9, MS2-aptamer-containing sgRNAs (MS2-sgRNAs), and a CD63-MCP fusion protein for non-covalent RNP loading.
• Introduced mutations in MS2 aptamers and MCPs to alter binding affinity.
• Assessed Cas9 RNP loading, EV-mediated delivery, and gene-editing efficiency.

Main Results:

• Modulating MS2-MCP binding affinity significantly impacted functional RNP delivery.
• High-affinity interactions required photo-inducible release for efficient delivery, indicating cargo release as a bottleneck.
• Decreased binding affinity facilitated RNP delivery without additional release strategies, though very low affinity reduced loading and editing efficiency.
• Modifications to the MCP RNA-binding domain also affected functional delivery.

Conclusions:

• EVs are effective vehicles for functional Cas9-sgRNA complex delivery.
• Modulation of binding affinity is a viable strategy to enhance non-covalent RNP loading and delivery via EVs.
• Optimizing binding affinity can improve CRISPR/Cas9 delivery efficiency without necessitating complex cargo release mechanisms.