Characterization and engineering of highly efficient Cas12j genome editors

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• CRISPR-Cas enzymes are crucial for genome editing but large sizes hinder therapeutic delivery.
• Cas12j nucleases are compact alternatives but have limited editing efficiency.
• Novel Cas12j orthologs were identified from viral metagenomes with low native activity.

Purpose of the Study:

• To engineer hypercompact Cas12j nucleases for enhanced genome editing efficiency.
• To develop Cas12j-based tools for therapeutic applications with delivery constraints.
• To explore novel sequence contexts influencing Cas12j editing activity.

Main Methods:

• Identification and characterization of eight novel Cas12j orthologs.
• Engineering of T5 exonuclease-Cas12j fusions to enhance editing activity.
• Development of Cas12j-based adenine base editors by fusing with adenine deaminase.
• Assessment of genome-editing activity and base conversion in mammalian cells.

Main Results:

• Engineered Cas12j fusions demonstrated substantially enhanced genome-editing activity in mammalian cells.
• Editing efficiency reached levels comparable to established compact CRISPR-Cas editors.
• A previously unrecognized trinucleotide sequence context was identified as crucial for robust editing.
• Efficient A-to-G base conversion was achieved using Cas12j-based adenine base editors.

Conclusions:

• Engineering principles were established to convert compact Cas12j nucleases into efficient genome-editing platforms.
• Novel Cas12j orthologs and engineered fusions expand the CRISPR toolbox for therapeutic applications.
• The developed Cas12j systems are well-suited for delivery-constrained therapeutic applications due to their compact size and high efficiency.