Production of immune receptor knockout chickens via direct in vivo transfection of primordial germ cells.

The advancement of genetic engineering in chickens has enabled significant advancement in developmental biology, bioreactors, and disease resilience. The development of CRISPR/Cas9 genome engineering technology has further expanded the potential applications of genetic engineering in poultry. In this study we aimed to evaluate the efficacy of a direct in vivo transfection method, previously demonstrated to produce transgenic chickens, in generating gene knockout (KO) chickens. Specifically, we targeted the Interferon-α/β Receptor 1 (IFNAR1) and Interleukin 1 receptor, type I (IL1R1), both critical pathways in the inflammatory and antiviral responses. We designed guide RNAs targeting the genes and validated their efficiency in vivo via microinjection into the developing embryos. PCR analysis confirmed the presence of gene deletions in chimeric roosters, which were subsequently bred to produce G1 germline heterozygote KO offspring. Homozygous KO chickens were generated and subjected to phenotypic and functional analyses. Our results demonstrated successful generation of functional knockouts of both IFNAR1 and IL1R1 using a direct in vivo transfection. Overall, this study demonstrates that direct in vivo transfection provides a robust and predictable method for generating KO chickens, facilitating further research into avian immune responses and the development of antiviral strategies.