Engineering CjCas9 for Efficient Base Editing and Prime Editing.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biomedical Research

Background:

• CRISPR-Cas9 gene therapy faces challenges with large Cas9 protein size, limiting adeno-associated virus vector packaging.
• Streptococcus pyogenes Cas9 is commonly used but difficult to deliver efficiently.
• Campylobacter jejuni Cas9 (CjCas9) is a smaller alternative, but its base editing (CjBE) and prime editing (CjPE) efficiencies are limited.

Purpose of the Study:

• To engineer enhanced CRISPR-Cas9 base editors (enCjBEs) and prime editors (enCjPEs) from CjCas9 for improved gene editing.
• To increase the efficiency and applicability of CjCas9-derived gene editing tools.
• To develop compact and efficient editors for biological research and gene therapy.

Main Methods:

• Engineered CjCas9 with P47K mutation to create enhanced cytosine base editors (enCjBEs) and adenine base editors (enCjABEs).
• Developed enhanced CjPE (enCjPE) using the CjCas9 P47K variant.
• Fused Sso7d and MS2 aptamer to create SsenCjPE, further optimized with hMLH1dn and MMLV RTaseΔRnH, and a D829R mutation for SsenCjPE-M2.

Main Results:

• Achieved robust C-to-T conversion (70%) with enCjBEs and A-to-G conversion (76%) with enCjABEs.
• enCjPE showed a 17-fold increase in editing efficiency at the PRNP site compared to wild-type CjPE.
• SsenCjPE achieved 12% average editing efficiency (24-fold increase), and SsenCjPE-M2 showed a 61-fold increase at the PRNP site.

Conclusions:

• Engineered CjCas9 variants (enCjBEs, enCjABEs, enCjPEs, SsenCjPEs, SsenCjPE-M2) offer compact and highly efficient gene editing solutions.
• These novel editors significantly enhance base and prime editing capabilities.
• The developed tools hold promise for advancing gene therapy and biological research applications.