Viral Delivery of Compact CRISPR-Cas12f for Gene Editing Applications

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• In vivo gene editing for genetic conditions necessitates effective delivery of CRISPR-Cas systems to target cells and organs.
• Clinical progress in gene editing includes ex vivo therapies and in vivo liver delivery via lipid nanoparticles.
• Adeno-associated virus (AAV) vectors are versatile for genetic material delivery across numerous organs, but large CRISPR-Cas systems pose packaging challenges.

Purpose of the Study:

• To develop a compact CRISPR-Cas gene editing system suitable for AAV packaging and in vivo applications.
• To demonstrate the efficacy and safety of a miniature CRISPR-Cas12f system for therapeutic gene editing.
• To expand the potential of CRISPR gene editing for diverse genetic targets and organs.

Main Methods:

• Engineered a miniature CRISPR-Cas12f system with expanded targeting capabilities.
• Developed a single AAV vector encoding both Cas12f nuclease and guide RNAs for four therapeutic targets.
• Validated gene editing efficiency in various cell lines, patient fibroblasts, and primary hepatocytes.
• Assessed off-target editing to evaluate the safety profile of the developed system.

Main Results:

• Demonstrated efficient CRISPR gene editing using the miniature Cas12f system delivered via AAV.
• Successfully encoded and delivered the Cas12f system and guide RNAs within a single AAV particle.
• Achieved gene editing in multiple cell types, including patient-derived cells and primary hepatocytes.
• Confirmed low levels of off-target editing, indicating a favorable safety profile.

Conclusions:

• The miniature CRISPR-Cas12f system packaged in AAV represents a significant advancement for in vivo gene editing.
• This technology facilitates efficient and targeted gene editing across diverse cell types and organs.
• The findings pave the way for broader applications of CRISPR editing in treating genetic diseases.