维持人胚胎干细胞多能性顺式作用元件的筛选  

Screening of cis-acting elements for maintaining pluripotency of human embryonic stem cells

作　　者：孙梦瑶 刘思琪 周凡琦 马艳妮[1] 余佳[1] SUN Meng-yao;LIU Si-qi;ZHOU Fan-qi;MA Yan-ni;YU Jia(State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院,医学分子生物学国家重点实验室,北京100005

出　　处：《基础医学与临床》2021年第5期636-640,共5页Basic and Clinical Medicine

基　　金：国家自然科学基金(81970103)。

摘　　要：目的在人胚胎干细胞(hESCs)中建立一种可行的CRISPR文库筛选方法,筛选参与维持hESCs多能性的顺式作用元件。方法将诱导型Cas9蛋白表达元件插入hESC基因组中,构建诱导型Cas9稳定表达株(iCas9-hESC)。通过分析已发表的hESCs中的染色质互作信息,确定可能参与多能性基因调控的顺式作用元件,并以此来构建双gRNA CRISPR敲除文库。使用CIRSPR文库在低感染复数情况下感染iCas9-hESC,诱导Cas9蛋白表达,对细胞进行基因编辑。以OCT4的表达水平作为衡量多能性的标准,对基因编辑后的细胞进行OCT4的流式抗体染色与分析,确定顺式元件敲除后是否影响到hESC的多能性。结果成功构建iCas9-hESC,且该细胞仍保持多能性状态。构建顺式元件的CRISPR敲除文库,成功感染目的细胞。对文库感染后的细胞进行OCT4表达水平的流式分析,发现基因编辑后的细胞确实存在多能性下降的部分群体。结论建立了在hESC中通过CRISPR文库筛选参与维持多能性顺式作用元件的可行性方案。Objective To establish an inducible CRISPR library screening method for human embryonic stem cells(hESCs)to screen the cis-acting elements involed in the maintainance the pluripotency of hESCs.Methods The inducible Cas9 expression elements were inserted into the hESC genome to construct an inducible Cas9 stable expression strain(iCas9-hESC).Analyze the published chromatin interaction information in hESCs to identify the cis-acting elements that may be involved in the regulation of pluripotency genes,then to construct a double gRNA CRISPR knockout library.The CIRSPR library was used to infect iCas9-hESC at low multiplicity of infection followed by induction of Cas9 expression,and gene editing of the cells.Using the expression level of OCT4 as a standard to measure pluripotency,the cells after gene editing were stained with OCT4 and analyzed by flow cytometry to determine whether the knockout of cis-elements would affect the pluripotency of hESCs.Results The iCas9-hESC was successfully constructed,and the cells still maintained a pluripotent state.Construct a CRISPR knockout library of ciselementsand successfully infect the target cells.Flow cytometric analysis of the expression level of OCT4 was performed on the cells after the library infection,and it was found that there were indeed some populations with decreased pluripotency in the cells after gene editing.Conclusions A feasible scheme for screening cis-acting elements involved in maintaining pluripotency in hESCs through CRISPR library is established.

关 键 词：人胚胎干细胞 多能性 顺式元件 CRISPR文库 

分 类 号：Q523[生物学—生物化学]