A Meta-Analysis of Animal Experiments on CRISPR-Cas9 Gene Editing for Hereditary Blood Disorders: Efficacy Assessment and Challenges in Hematopoietic System Reconstitution

Background: Hereditary blood disorders lack curative therapies. As an emerging therapeutic strategy, the efficacy and safety of CRISPR-Cas9 gene editing technology in preclinical animal models require systematic integration and evaluation. This study, through a meta-analysis, aims to comprehensively evaluate the editing efficiency, therapeutic efficacy, and impact on hematopoietic system reconstitution of CRISPR-Cas9 in animal models, in order to clarify its potential for clinical application and the existing challenges. Methods: Following the PRISMA guidelines, we systematically searched the PubMed, Embase, Cochrane Library, Web of Science, CNKI, and Wanfang databases for animal studies on hereditary diseases involving CRISPR-Cas9 gene editing technology, up to September 2024. Data on gene editing efficiency, induced insertions and deletions (InDels), cellular markers (hCD45 +, percentage of CD235a + erythrocytes, and CD3 + immune cell levels), and lymphocyte subsets in the bone marrow and spleen (CD34 +, CD19 +, CD3 + ) were extracted. The SYRCLE tool was used to assess the risk of bias in the animal experiments. Statistical analyses were performed using RevMan 5.3 software, and the effect sizes were pooled using either a fixed effect or random effects model based on the results of heterogeneity testing (I 2 statistic and 𝜒 2 test). Results: Finally, 18 studies on gene editing for hereditary blood disorders (mainly focusing on sickle cell disease, β thalassemia, and some immunodeficiency disorders) were included. The core target genes edited mainly included BCL11A, the hemoglobin β chain gene (HBB), and the fetal hemoglobin genes (HBG1/HBG2). The efficiency of input edited cells was significantly higher than that of endpoint edited cells (SMD = 1.71, 95% CI: 0.91-2.51; P < 0.0001). The overall induced InDel frequency in the edited group (EG) was significantly lower than that in the control group (CG) (SMD = −0.42, 95% CI: −0.70 to −0.13; P = 0.005). In the spleen, the proportion of hCD45 + cells in the EG was significantly lower than that in the CG (SMD = −2.73, 95% CI: −4.88 to −0.57; P = 0.01). The percentage of erythrocytes in the EG was significantly higher than that in the CG (SMD = 3.01, 95% CI: −1.23 to 4.80; P = 0.0009). The CD3 + level in the bone marrow was significantly higher in the EG than in the CG (SMD = −1.44, 95% CI: −2.74 to −0.14; P = 0.03), and the CD3 + level in the spleen was also significantly higher in the EG than in the CG (SMD = −5.91, 95% CI: −10.31 to −1.50; P = 0.009). However, the proportion of hCD45 + in the bone marrow, the proportion of CD235a + in the bone marrow, β globin levels, as well as CD34 + and CD19 + levels in the bone marrow showed no statistically significant differences between the two groups (P > 0.05). Conclusion: CRISPR-Cas9 has demonstrated high initial editing efficiency in animal models of inherited hematological disorders and has successfully produced phenotypic improvements, manifested as an increased percentage of red blood cells in mice after transplantation, suggesting its potential therapeutic prospects for correcting the anemic phenotype. However, from in vitro editing to stable in vivo functional erythropoiesis, multiple biological hurdles-including homing, long-term engraftment, and stress adaptation-must be overcome, and the full efficacy remains to be confirmed by longer-term and more in-depth in vivo functional studies.