CRISPR/Cas9 is a powerful gene-editing tool that has revolutionized genetics and molecular biology research. It enables precise manipulation of DNA sequences within the genome, allowing researchers to create small animal models with specific gene modifications. CRISPR/Cas9-based gene editing in small animals typically involves the delivery of Cas9 nuclease and a guide RNA (gRNA) to the target cells, where the gRNA directs Cas9 to a specific genomic location to introduce a double-strand break, which is then repaired by cellular repair mechanisms, potentially resulting in gene disruption, deletion, or insertion.

Various transfection approaches can be employed to deliver CRISPR/Cas9 components in small animals, including:

1. Viral vectors: Adeno-associated virus (AAV) and lentivirus are commonly used viral vectors for delivering CRISPR/Cas9 components in small animals. AAV is generally preferred for its low immunogenicity and broad tropism. However, the limited packaging capacity of AAV may require splitting the Cas9 and gRNA into separate vectors. Lentiviruses offer larger packaging capacity, but carry a risk of insertional mutagenesis.
2. Electroporation: Electroporation can be used for in vivo delivery of CRISPR/Cas9 components. This approach involves injecting the Cas9 protein or mRNA along with the gRNA into the target tissue, followed by the application of an electric field to facilitate the uptake of these molecules into cells. In utero electroporation is a popular method for generating genetically modified small animals, particularly in the brain of developing embryos.
3. Microinjection: Microinjection of CRISPR/Cas9 components directly into the pronucleus of a fertilized egg or the cytoplasm of a zygote is a widely used approach for generating genetically modified small animals, such as mice and rats. The injected embryos are then implanted into a surrogate mother to develop.
4. Nanoparticle-mediated delivery: Nanoparticles, such as lipid-based nanoparticles, polymer-based nanoparticles, or inorganic nanoparticles, can be used to encapsulate or complex with CRISPR/Cas9 components for delivery in small animals. This approach is attractive due to its versatility, biocompatibility, and potential for targeted delivery.
5. Sonoporation and microbubble-assisted transfection: Ultrasound-mediated delivery of CRISPR/Cas9 components using microbubbles can be employed for gene editing in small animals. This non-invasive approach allows for targeted delivery of the CRISPR/Cas9 system to specific tissues or organs.

Each of these approaches has its advantages and limitations in terms of efficiency, specificity, invasiveness, and potential side effects. Researchers must carefully consider the most appropriate method for their specific experimental goals and the species of small animals being used. Regardless of the delivery method, optimizing parameters such as gRNA design, Cas9 dosage, and delivery timing is crucial for achieving efficient and precise gene editing in small animals.