CRISPR/Cas9-mediated β-globin gene knockout in rabbits recapitulates human β-thalassemia

Abstract

β-thalassemia, an autosomal recessive blood disorder that reduces the production of hemoglobin, is majorly caused by the point mutation of the HBB gene resulting in reduced or absent β-globin chains of the hemoglobin tetramer. Animal models recapitulating both the phenotype and genotype of human disease are valuable in the exploration of pathophysiology and for in vivo evaluation of novel therapeutic treatments. The docile temperament, short vital cycles, and low cost of rabbits make them an attractive animal model. However, β-thalassemia rabbit models are currently unavailable. Here, using CRISPR/Cas9-mediated genome editing, we point mutated the rabbit β-globin gene HBB2 with high efficiency and generated a β-thalassemia rabbit model. Hematological and histological analyses demonstrated that the genotypic mosaic F0 displayed a mild phenotype of anemia, and the heterozygous F1 exhibited typical characteristics of β-thalassemia. Whole-blood transcriptome analysis revealed that the gene expression was altered in HBB2-targeted when compared with WT rabbits. And the highly expressed genes in HBB2-targeted rabbits were enriched in lipid and iron metabolism, innate immunity, and hematopoietic processes. In conclusion, using CRISPR-mediated HBB2 knockout, we have created a β-thalassemia rabbit model that accurately recapitulates the human disease phenotype. We believe this tool will be valuable in advancing the investigation of pathogenesis and novel therapeutic targets of β-thalassemia and associated complications.

Keywords: CRISPR/Cas; animal model; gene therapy; genetic disease; hemoglobin; rabbit; β-thalassemia.

Figure 1

CRISPR/Cas9-mediated gene targeting of rabbit HBB2.A, genomic structure of the β-globin gene cluster and schematic diagram of the Cas9/gRNA targeting sites in rabbit HBB2. Comparative genomics analysis showed that the β-globin gene families are highly conserved between rabbit and human, which contain only one adult β-globin gene (highlighted in red box); Two gRNAs were designed targeting exon1 and exon2 of rabbit HBB2, gRNA1 and gRNA2 are highlighted in red arrows. B, mutation detection of Cas9/gRNA1 and Cas9/gRNA2 in rabbit fetal fibroblasts by T7E1 cleavage assay. C, development of different stage of rabbit embryos with no injection (control group) and coinjected with Cas9/gRNA1 and Cas9/gRNA2 in vitro. D, mutation detection of Cas9/gRNA1 and Cas9/gRNA2 with different concentration in rabbit embryos by T7E1 cleavage assay. E, mutagenic efficiency of embryo injection of Cas9/gRNA detected by Sanger sequencing.

Figure 2

Generation of HBB2-targeted rabbit via embryo injection of Cas9/gRNA.A, summary of embryo injection and transplantation. B, genotype of HBB2-targeted rabbits detected by T-A cloning and Sanger sequencing. C, photographs of HBB2-targeted rabbits F0-5, F0-6, F0-7, and F0-8 at 1 month old. D, HBB2 mutant proration in the skin and blood of the genotypic mosaic F0 rabbits. E. T7E1 assays of the predicated off-target sites (Fig. S2).

Figure 3

Heritability and phenotype of HBB2-targeted rabbits.A, T7E1 cleavage assay for mutant HBB2 detection in F1 HBB2-targeted rabbits. B, hematologic features of HBB2-targeted rabbits. Hematological values are expressed as means+ SD. Hb, hemoglobin concentration; HCT, hematocrit; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; RBC, red blood cells; RDW, red cell distribution width; Retic, reticulocyte count. C, peripheral blood smears of WT and HBB2-targeted rabbits. Scale bars: 10 μm. Iron staining of the spleen, liver, and kidney in WT and HBB2-targeted rabbits. Scale bars: 50 μm.

Figure 4

The whole-blood transcriptome profiles of WT and HBB2-targeted rabbits.A, the expression level of genes in WT and HBB2-targeted F0 and F1 rabbits. B, logs-fold change of the top ten highly expressed genes in HBB2-targeted F0 and F1 rabbits. C, go classification of the genes expressed in the blood of HBB2-targeted rabbits.

Figure 5

Differential gene expression pattern in the blood of HBB2-targeted rabbits.A, the cluster heat map of the top200 genes with the highest variance in WT and HBB2-targeted rabbits. B, volcano plots of differential gene expression level. The padj <0.05 (FDR < 0.1), HBB2-targeted-F0 (red dots), HBB2-targeted F1 (blue dots). C, the differential expression of genes related to hematopoiesis. D, the differential expression of genes related to innate immune. E, the differential expression of genes related to various blood disorders.