Abstract: 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 ciselements and 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.

Key words: human embryonic stem cell, pluripotency, cis-elements, CRISPR library