DNA-free gene editing in vivo
The zebrafish (Danio rerio) is a preferred model system for biomedical research due to its high sequence homology with humans. This model is relatively easy to handle in the lab and is a genetically tractable system that undergoes rapid external development, making it ideal to study early developmental events. Being that they are highly amenable to genetic manipulation, and the molecular tools necessary to perform the targeted genomic editing are already available, zebrafish are an increasingly popular choice for targeted genomic modification using the CRISPR-Cas9 gene editing system.
The CRISPR-Cas9 system requires exogenous Cas9 nuclease to be delivered into the cell, which can be accomplished through transfection of an expression plasmid, mRNA or protein, or through transduction with lentiviral particles. DNA-based Cas9 constructs, while appropriate for many applications, may result in unwanted integration events. Lentiviral delivery results in integration of the Cas9 expression cassette into the cell’s genome, and transfection of a Cas9 plasmid may result in the insertion of vector sequence at the site of the double-strand break. The use of Cas9 mRNA or protein avoids any unwanted integration, and when used in combination with synthetic crRNA and tracrRNA, results in a completely DNA-free gene editing system.
Gene Editing in Zebrafish via microinjection with CRISPR-Cas9 reagents
In a recently published applications note and scientific poster, we have demonstrated successful gene editing using DNA-free CRISPR-Cas9 reagents for gene knockout in zebrafish. Zebrafish embryos from a stable transgenic line were injected with Cas9 nuclease mRNA, synthetic tracrRNA, and crRNA designed to target GFP. A mismatch detection assay confirmed efficient gene editing, and successful functional protein knockout was confirmed by loss of GFP fluorescence.
These new resources provide guidance on how to perform gene knockout in Zebrafish and demonstrate the following findings:
- DNA-free gene editing using synthetic crRNA:tracrRNA is highly efficient and easy to use
- The removal of CRISPR-Cas9 DNA components increase experimental confidence while reducing potential off-targets
- Zebrafish are an amenable organism for DNA-free genome engineering experiments