In vivo genome editing with a novel Cj4Cas9

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 systems are powerful genome editing tools.
• The search for compact and efficient CRISPR-Cas9 variants is ongoing.
• Natural CRISPR systems offer diverse properties for gene editing applications.

Purpose of the Study:

• To identify novel, compact CRISPR-Cas9 systems from natural sources.
• To characterize and engineer these systems for enhanced genome editing capabilities.
• To evaluate their potential in fundamental research and therapeutic applications.

Main Methods:

• Screening of 11 CjCas9 orthologs using a GFP activation assay.
• In vivo validation in mouse models (zygotes and liver).
• Engineering of Cj4Cas9 to create a high-activity variant (enCj4Cas9).

Main Results:

• Identified seven active CjCas9 nucleases, with Cj4Cas9 being notably compact (985 amino acids) and having a 5'-NNNGRY-3' PAM.
• Demonstrated efficient disruption of the Tyr gene in mouse zygotes, causing an albino phenotype.
• Showcased in vivo genome editing of the Pcsk9 gene in mouse liver via AAV8 delivery, reducing cholesterol levels.
• Engineered enCj4Cas9 with increased nuclease activity and a simplified N3GG PAM, expanding targeting scope.

Conclusions:

• Cj4Cas9 is a promising compact genome editing tool with unique PAM specificity.
• Engineered variants like enCj4Cas9 significantly enhance editing efficiency and expand targeting range.
• These CRISPR-Cas9 systems hold potential for both basic research and therapeutic development.