Development and characterization of a CRISPR/Cas9-mediated RAG1 knockout chicken model lacking mature B and T cells

Abstract

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 (RAG1) 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 RAG1 ^+/- and RAG1 ^-/- 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 RAG1 ^+/- chickens, lymphatic organs in 3-week old RAG1 ^-/- chickens were severely damaged. Furthermore, relative to WT chickens, RAG1^+/- and RAG1^-/- birds had reduced serum Igs, fewer mature CD4⁺ and CD8⁺ T lymphocytes. Furthermore, BCR-mediated B cell activation in RAG1 ^+/- chickens was insufficient, leading to decreased expression of the activation-induced deaminase (AID) gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of RAG1 in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of RAG1 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.

Keywords: B cell; B cell receptor; CRISPR/Cas9; RAG1 knockout; T cell; avian immunology; embryonic stage; immunodeficient chicken.

Figure 1

Generation of RAG1 knockout chickens via primordial germ cell (PGC)-mediated germline transmission. (A) CRISPR/Cas9 targeting exon 1 of RAG1 (RAG1 #2) with a donor plasmid containing tdTomato, driven by the CMV promoter to disrupt RAG1 expression and schematic structure of chicken RAG1 protein. In the schematic diagram of the RAG1 protein, numbers indicate amino acids number. (B) Expression of tdTomato in chicken PGCs. Scale bars = 100μm. (C) Genomic DNA analysis of targeted genome integration of 5’ and 3’ junctions in chicken PGCs using integration-specific primer sets, and sequencing results following TA cloning of amplicon. (D) Production of RAG1 knockout chickens, distinguished by red fluorescence. (E, F) Representative results of integration-specific PCR amplification of genomic DNA from WT, RAG1 ^+/- and RAG1 ^-/- progeny, and results of sequencing the 5’ and 3’ junctions.

Figure 2

Expression profile of RAG1 in lymphoid organs and decreased B cell population in RAG1 ^+/- and RAG1 ^-/- embryonic bursa. Validation of RAG1 mRNA expression by RT-qPCR analyses in the primary lymphoid organs [bursa (A) and thymus (C)], and the secondary lymphoid organ [spleen (B)]. Relative expression of RAG1 was calculated after normalization to GAPDH and E8 control samples, and the significance of differences among groups was evaluated by one-way ANOVA; *P < 0.05, ***P < 0.001, ****P < 0.0001. Expression of the B cell lineage marker (Bu-1) in E16 (D) and E18 (E) embryonic bursa was examined by immunohistochemistry.

Figure 3

RAG1 deficiency impairs B cell development in embryonic bursa, as demonstrated by reduced sIgM expression and impaired CXCR4 and CXCL12 interaction in RAG1 ^+/- and RAG1 ^-/- embryonic bursa. (A) Expression of each immunoglobulin M domain in total bursa cDNA samples from each group, as determined by RT-PCR. (B) The proportions of surface IgM (sIgM) -positive cells in E18 bursa from the WT, RAG1 ^+/-, and RAG1 ^-/- groups were determined by immunostaining with IgM. Relative expression levels of RAG1, immunoglobulin light chain (Ig Light), B cell development-related genes, and bursa environment-related genes were calculated after normalization to GAPDH and B cell specific gene BAFF at E16 (C) and E18 (D), and the significance of differences among groups was determined by one-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance.

Figure 4

Impaired development of embryonic B cell bursa follicle. (A) Histological images of WT, RAG1 ^+/-, and RAG1 ^-/- E18 bursa. The number of follicles (B) and diameter (C) of bursa follicles within 1-fold of bursa. The significance of differences among groups was calculated by one-way ANOVA; ns, no significance, **P < 0.01 and ****P < 0.0001. (D) Embryonic bursa sections were stained with desmin at E18 to identify bursa follicle compartmentalization.

Figure 5

Impaired development of lymphoid organs in RAG1 knockout chickens. Comparison of WT, RAG1 ^+/-, and RAG1 ^-/- chicken body weight at 1 week (A) and 3 weeks (C). The significance of differences among groups was calculated by one-way ANOVA; *P < 0.05, ***P < 0.001, ****P < 0.0001. Morphology and histological images of WT, RAG1 ^+/-, and RAG1 ^-/- chicken lymphoid organs at 1 week (B) and 3 weeks (D). Asterisks indicate gross lesions. Prepared sections were stained with hematoxylin and eosin (H&E). Scale bars = 1 cm for bright field images, and 50 μm for H&E-stained images. (E) Diagram of immunoglobulin heavy chain (top) and light chain (bottom) loci in germline and rearranged DNA. PCR analysis of PBMC gDNA from 1-week old WT, RAG1 ^+/-, and RAG1 ^-/- chickens using specific primers (VH-F_JC-R and VL-F_VL-R) targeting rearranged immunoglobulin DNA. ns, no significance.

Figure 6

Immune cell populations and quantification of secreted immunoglobulins in RAG1 knockout chickens. Representative flow cytometry analysis of B and T cell subpopulations in the bursa (A), and thymus (B) of 3-week-old chickens. Statistical analysis was performed on Bu-1 and IgM positive cells in the bursa as well as CD4⁺ and CD8⁺ single-positive (SP) T cells, and the CD4⁺CD8⁺ double-positive (DP) T cell lymphocyte subpopulation in the thymus. Serum IgM, IgY, and IgA levels in 1-week (C) and 3-week (D) -old WT, RAG1 ^+/-, and RAG1 ^-/- chickens, as determined by ELISA. Each dot represents an individual chicken. The significance of differences among groups was assessed by one-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance.

Figure 7

Survival rate of WT, RAG1 ^+/-, and RAG1 ^-/- chickens in conventional breeding condition. (A) A curve showing the survival rate after hatch. (B) The disrupted organs (liver and spleen) of RAG1 ^-/- chickens over time.

Figure 8

Immature B cell development caused by RAG1 haploinsufficiency affects immunoglobulin gene conversion. (A) Verification of Bu-1 and GAPDH expression in MACS sorted Bu-1 positive cells for RT-PCR. (B) Relative expression of B cell development and BCR downstream signal related genes in Bu-1-positive sorted B cells from the bursa of 3-week-old WT and RAG1 ^+/- chickens, calculated after normalization to GAPDH and WT samples. The significance of differences between groups was assessed by Student t-test; *P < 0.05. (C) Analysis of the proportion of clones containing threonine codons in the region encoding the CDR3 domain of the immunoglobulin heavy chain in PBMC gDNA from 3-week-old WT and RAG1^+/- chickens, and amino acid sequence alignment of CDR3 domains based on sequencing analysis. Red box, threonine within the CDR3 region. ns, no significance.