Efficient CRISPR/Cas9-mediated knockin of reporter genes in rats at ROSA26 by pronuclear microinjection.

The genetic modification of rats is a key technology for advancing biomedical research on human diseases. CRISPR/Cas9-mediated genome editing enables the generation of knockout rats in a single step, without the need for embryonic stem cells, by directly injecting genome editing components into zygotes. This simplifies the process, reduces costs, and accelerates gene function analysis in rats. However, the insertion of a gene cassette into a target site has remained inefficient, limiting the generation of knockin (KI) rats. To overcome this issue, we developed an optimized method that covers the entire process from zygote harvesting with superovulation to timed microinjection, ensuring the consistent generation of KI rats. We successfully generated four different fluorescent reporter lines at the ROSA26 locus in rats. Our study provides detailed, step-by-step protocols for donor vector design, zygote collection, microinjection, founder screening, and cryopreservation in rats.