Lentiviral Capsid-Mediated Streptococcus pyogenes Cas9 Ribonucleoprotein Delivery for Efficient and Safe Multiplex...

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Transient expression of CRISPR-Cas9 is crucial for minimizing off-target mutations and immune reactions.
• Electroporation of Cas9 ribonucleoproteins (RNPs) is a standard delivery method, but fusion strategies with viral proteins can reduce assembly efficiency.
• Virus-like particles (VLPs) offer a promising alternative for delivering gene editing tools.

Purpose of the Study:

• To investigate the efficient generation of highly active Streptococcus pyogenes Cas9 (SpCas9) RNP VLPs using aptamer/aptamer-binding protein (ABP) interactions.
• To evaluate the safety and efficiency of SpCas9 RNP VLPs compared to traditional electroporation methods.
• To assess the suitability of Cas9 VLPs for multiplex genome editing.

Main Methods:

• Optimizing aptamer locations and types for single-guide RNA modification in SpCas9 RNP VLP construction.
• Comparing off-target mutations between VLP-delivered SpCas9 and electroporation-delivered SpCas9 RNPs.
• Preparing and testing VLPs containing Cas9 from different species for single and multiplex gene targeting.

Main Results:

• Highly active SpCas9 RNP VLPs were generated efficiently through aptamer/ABP interactions.
• VLP-delivered SpCas9 resulted in fewer off-target insertions and deletions than electroporation.
• Multiplex-target VLPs demonstrated superior efficiency for simultaneous gene editing compared to single-target VLP combinations.

Conclusions:

• Optimized Cas9 RNP VLPs provide a safe, efficient, and flexible platform for genome editing.
• Cas9 VLPs are particularly well-suited for multiplex genome editing applications.
• This VLP system enhances safety by reducing off-target effects and offers improved efficiency for complex editing tasks.