Short term but highly efficient Cas9 expression mediated by excisional system using adenovirus vector and Cre

Area of Science:

• Molecular Biology
• Gene Therapy
• Virology

Background:

• CRISPR/Cas9 is a common gene engineering tool but faces challenges in efficiency and off-target effects.
• Persistent Cas9 expression from traditional adenovirus vectors can be problematic for gene therapy.

Purpose of the Study:

• To develop a novel Cas9 transduction system, Exci-Cas9, to improve genome editing efficiency and control expression duration.
• To evaluate Exci-Cas9 for targeting persistent viral infections, exemplified by hepatitis B virus.

Main Methods:

• Developed Exci-Cas9 using an adenovirus vector (AdV) where Cas9 is expressed from a circular molecule excised by Cre recombinase.
• Compared Exci-Cas9 to direct AdV-mediated Cas9 expression (Avec) for hepatitis B virus genome disruption.
• Investigated cell- or tissue-specific genome editing using Exci-Cas9 with cell-specific Cre promoters.

Main Results:

• Exci-Cas9 significantly shortened Cas9 expression periods compared to standard AdV vectors.
• Achieved high efficiencies (80-90%) of hepatitis B virus genome disruption with Exci-Cas9, comparable to Avec.
• Demonstrated the capability of Exci-Cas9 for cell- or tissue-specific genome editing.

Conclusions:

• Exci-Cas9 offers a solution to the persistent Cas9 expression issue in genome editing.
• This novel system holds potential for eliminating long-term DNA viruses and advancing targeted gene therapies.