Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The Cas9 enzyme is a key tool for genome editing.
• Large Cas9 variants limit therapeutic applications due to packaging constraints in viral vectors.

Purpose of the Study:

• To characterize the compact Cas9 from Campylobacter jejuni (CjCas9).
• To engineer an improved CjCas9 variant for enhanced genome editing.
• To evaluate the potential of engineered CjCas9 for therapeutic applications.

Main Methods:

• Biochemical characterization of CjCas9.
• Engineering of a CjCas9 variant (enCjCas9) with altered properties.
• In vitro and in human cell assays to assess cleavage activity and targeting range.
• Fusion of enCjCas9 nickase with cytosine deaminase for base editing.

Main Results:

• CjCas9 exhibits a novel preference for the N3VRYAC protospacer adjacent motif.
• Engineered enCjCas9 shows enhanced cleavage activity and a broader targeting range than wild-type CjCas9.
• enCjCas9 nickase enables efficient C-to-T base conversions in human cells when fused with cytosine deaminase.

Conclusions:

• Compact CjCas9 offers advantages over larger Cas9 orthologs for gene editing.
• Engineered enCjCas9 expands the CRISPR-Cas toolkit for therapeutic genome engineering.
• The developed enCjCas9 system holds promise for precise and efficient therapeutic genome editing.