Engineered Cas12j-8 is a Versatile Platform for Multiplexed Genome Modulation in Mammalian Cells

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Cas12j-8 is a compact CRISPR-Cas nuclease with a '5-TTN-3' PAM.
• Its low efficiency in mammalian cells hinders therapeutic applications.
• Structure-guided mutagenesis can improve DNA binding and editing efficiency.

Purpose of the Study:

• To engineer Cas12j-8 for enhanced gene editing efficiency in mammalian cells.
• To develop base editors (CBEs and ABEs) using the engineered Cas12j-8.
• To evaluate the multiplexing capability and therapeutic potential of engineered Cas12j-8.

Main Methods:

• Structure-guided mutagenesis was used to create a quintuple mutant (enCas12j-8).
• Engineered Cas12j-8 was utilized to develop cytosine and adenine base editors.
• Multiplexed editing of three genomic loci and induction of exon skipping were performed.

Main Results:

• Engineered Cas12j-8 showed robust on-target editing efficiency comparable to LbCas12a with low off-target effects.
• Base editors achieved significant C-to-T and A-to-G conversion efficiencies.
• Simultaneous multiplexed editing and therapeutic exon skipping were successfully demonstrated.

Conclusions:

• Engineered Cas12j-8 is a versatile and high-precision tool for genome engineering.
• It offers efficient delivery, multiplexing, and compatibility with diverse editing modalities.
• This technology holds significant potential for therapeutic genome modulation.