Development and characterization of a CRISPR/Cas9-mediated knockout chicken model lacking mature B and T cells.

Although birds have been used historically as a model animal for immunological research, resulting in remarkable achievements, immune cell development in birds themselves has yet to be fully elucidated. In this study, we firstly generated an immunodeficient chicken model using a CRISPR/Cas9-mediated recombination activating gene 1 () knockout, to investigate avian-specific immune cell development. Unlike previously reported immunoglobulin (Ig) heavy chain knockout chickens, the proportion and development of B cells in both and embryos were significantly impaired during B cell proliferation (embryonic day 16 to 18). Our findings indicate that, this is likely due to disordered B cell receptor (BCR)-mediated signaling and interaction of CXC motif chemokine receptor (CXCR4) with CXCL12, resulting from disrupted Ig V(D)J recombination at the embryonic stage. Histological analysis after hatching showed that, unlike wild-type (WT) and chickens, lymphatic organs in 3-week old chickens were severely damaged. Furthermore, relative to WT chickens, and birds had reduced serum Igs, fewer mature CD4 and CD8 T lymphocytes. Furthermore, BCR-mediated B cell activation in chickens was insufficient, leading to decreased expression of the activation-induced deaminase () gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of during immune cell development. This model will provide ongoing insights for understanding chicken immune system development and applied in the fields of immunology and biomedical science.