A New Approach for Large DNA Insertions Using rAAV and CRISPR
Inserting large DNA fragments into the genome presents challenges in efficiency and precision. Traditional plasmid-based approaches require microinjection, which is time-consuming, inefficient, and results in low success rates in animal models. This method, utilizing recombinant AAV (rAAV) alongside CRISPR, offers a more effective alternative.
Key Features of This Method
- Improved Efficiency – Electroporation of rAAV provides better success rates compared to microinjection.
- Scalability – Previously, projects required 6-8 microinjection sessions; this method typically requires only 1-2.
- Expanded Insert Size – Uses multiple rAAV vectors to insert up to 9 kb of genetic material, surpassing the 4.7 kb limit of a single rAAV.
- Consistent Results – Reduces variability by avoiding random integration and concatemer formation.
- Multiplexing Capabilities – Allows for simultaneous modification of multiple targets.
This method provides a scalable approach to verifying transgene integration, ensuring that edits are made as intended and minimizing the risk of unexpected genomic alterations.
-Monica F. Sentmanat, Lead Author
How It Works
Electroporation-Based Delivery
- rAAV donors are incubated with embryos, removing the need for microinjection.
- CRISPR components are electroporated, improving targeting precision.
Sequential Integration with Guide RNAs
- The first rAAV inserts a portion of the DNA and introduces a guide RNA cut site.
- A second rAAV inserts additional DNA, followed by a third, allowing integration of up to 9 kb.
Genotyping with Long-Read Sequencing
- This approach enables precise mapping of insertions, off-target effects, and structural variations.
Hear from Xiaoxia Cui: Insights on This Technique
Xiaoxia Cui, PhD, the director of our lab, discusses the rationale behind this approach, its applications, and how it enhances genotyping accuracy.
Interested in starting a project? Contact us.