应用CRISPR/Cas9技术筛选γ珠蛋白基因转录抑制因子BCL11A的结合位点及功能验证

Recently, the repressing transcription factor BCL11A was found as a critical regulator of γ- to β-globin gene switching, with a binding site within a 3.5kb region at the 5' upstream of δ-globin gene. However, the precise binding site of BCL11A is unclear. CRISPR/Cas9 system as a genome editing tool developed quickly in recent three years, and has the advantages of high through-put and high accuracy for functional screening of genes. In this project, all feasible sgRNAs within the 3.5kb region at the 5' upstream of δ-globin gene will be designed, and lentivirus sgRNA library will be constructed. CD34+ haemopoietic stem cells will be transduced with sgRNA lentivirus followed by induced erythroid differentiation. Populations of cells with high and low expression of HbF will be sorted by FACS. sgRNAs related to high expression of HbF will be identified by deep sequencing, and the precise binding site of BCL11A can be determined subsequently. Finally, the BCL11A-binding site specific mutation via CRISPR/Cas9 system will be performed to validate its function in repressing the expression of HbF. The results of this study will be benefit to further elucidate the molecular mechanism of γ- to β-globin gene switching, and provide new gene therapy target for β-thalassemia.

近年发现转录抑制因子BCL11A是γ→β珠蛋白基因转换过程中起关键作用的调节因子，其结合位点在δ珠蛋白基因5'端上游3.5kb范围内。然而，BCL11A的准确结合位点目前尚不清楚。CRISPR/Cas9系统最近3年迅速发展起来的基因编辑技术，在功能基因筛选方面具有通量大、准确性高的优势。本项目在δ珠蛋白基因5'上游3.5kb的区域内设计出所有可行的sgRNAs，构建sgRNAs慢病毒文库。CD34+造血干细胞感染sgRNAs慢病毒后向红系定向诱导分化。FACS分选出HbF高表达和低表达的细胞群，再用高通量测序鉴定出与HbF高表达相关的sgRNAs，从而推断出BCL11A的准确结合位点，并验证其抑制HbF表达的功能。本项目的研究结果将有助于进一步阐明γ→β珠蛋白基因转换过程的分子机制，同时为重型β-地贫的基因治疗提供了新的靶点。

重新激活成人体内已经沉默的γ-珠蛋白的表达从而治疗遗传性疾病β-地中海贫血是近年来的研究热点之一，以往研究报道HBD基因上游3.5kb区域与HbF的表达相关。我们设计了这一区域内所有的sgRNA并构建了CIRSPR质粒文库，经过NGS测序鉴定文库合格后构建了慢病毒文库。我们将CRISPR文库慢病毒感染HUDEP-2细胞并用流式分选出HbF表达最高和最低的细胞各10%，经过NGS测序获得了δ-珠蛋白基因上游3.5kb范围内所有sgRNA位点与HbF表达的关系。我们验证了与HbF表达相关的sgRNA位点，其中2个sgRNA（sgLIB6，sgLIB7）对HBG有促进作用，为β-地贫的基因治疗研究提供了新的靶点。本项目还对BCL11A红系特异增强子+58和BCL11A在γ-珠蛋白启动子区的结合位点进行了单独和联合编辑，在mRNA表达和蛋白表达水平，联合编辑对HBG的促进效果均明显优于单独编辑，这种联合编辑的策略为β-地贫的基因治疗提供了一个新的思路。