出生缺陷包括很多由基因突变所致的罕见病。对罕见病的研究有助于我们认识机体正常发育的机理。染色质重塑因子CHD7的杂合子突变导致一种严重的发育性疾病CHARGE综合征。前期研究结果提示CHD7在早期胚胎发育的神经胚形成阶段的神经上皮基因的激活不可或缺。申请人新建准确模拟患者突变的小鼠模型，并结合人大脑类器官实验模型的研究来阐明CHD7突变在神经胚时期的表型。以3D培养的人大脑类器官为细胞模型，利用分子生物学技术鉴定CHD7基因表达的动态调控。由于患者中CHD7突变均为杂合子突变，我们利用失活Cas9蛋白融合转录活化蛋白特异激活完整的野生型CHD7基因的表达，在人大脑类器官模型里测试对CHD7杂合突变所致表型的修复。申请课题取得的结果不仅将有助于我们认识染色质重塑因子在神经胚形成中的作用，还将帮助我们认识CHARGE综合征的致病机理和探索治疗新方法。

Birth defect are recurrent diseases for newborns, which are mostly caused by gene mutations. Research on these rare disease-causing genes help us to understand the general mechanism of development. Development is a very complex series of cell differentiation, which requires coordinated gene expression in each step. Chromatin remodelers are actively and directly involved in transcription, by controlling the accessibility of chromatin, the DNA template of transcription. Not surprisingly, mutations of chromatin remodelers lead to developmental diseases. Importantly, heterozygous mutation of chromatin remodeler CHD7 (chromo domain helicase DNA binding protein 7) leads to a severe developmental disorder named CHARGE syndrome. Gastrulation and neurulation are crucial period of early embryonic development, whereas dramatic programming of gene expression occurs. Preliminary data suggests that CHD7 play crucial role during neurulation by specifically activating neuroepthilia genes . We generate transgenic mouse lines to mimic Chd7 mutation found in CHARGE syndrome patients. We develop isogenic pluripotent stem cell lines carrying homozygous or heterozygous mutations of CHD7 gene, which can be differentiated into brain organoid, a new model system to study early development of human brain. Transgenic mouse and human brain organoid model will be combined to study the role of CHD7 during neurulation. We use 3D cultured organoid as the major cell system to study the molecular mechanism behind the function of CHD7 in neurulation. CHD7 is a typical haploidinsufficient gene, all known CHD7 mutations are non-exceptional heterozygous . We use dCas9 fused to transcriptional activator to upregulate the remaining intact CHD7 gene copy in order to recover the expression level of CHD7 in mutant cell and test whether this can rescue the phenotype of CHD7 heterozygous mutation using brain organoid model. Results from this project will not only enhance our understanding of the role of CHD7 during the neurulation step of early embryonic development, but also help us to understand the pathologic mechanism of CHARGE syndrome and to explore new potential therapy of this disease.