Establishment of human embryonic stem cell line with forkhead box G1 gene knockout by CRISPR/Cas9 / 第二军医大学学报

ABSTRACT

Objective To construct human embryonic stem cell (hESC) line with forkhead box G1 (FOXG1) gene knockout by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology, and to investigate the role of FOXG1 gene in the early neural induction of hESCs. Methods Two guide RNAs (gRNAs) were transfected to induce FOXG1 gene large fragment knockout in hESCs by CRISPR/Cas9 gene editing technology. FOXG1 gene knockout hESCs were confirmed by monoclonal screening, sequencing and Western blotting analysis. The expression of the key markers including paired box 6 (PAX6), sex-determining region Y-box 2 (SOX2) and orthodenticle homeobox 2 (OTX2) was detected by immunofluorescence staining and qRT-PCR in the early process of neural induction before and after FOXG1 gene knockout. Results hESCs with FOXG1 gene large fragment knockout were successfully obtained by CRISPR/Cas9 gene editing technology. The results of immunofluorescence staining and qRTPCR suggested that FOXG1 deletion did not significantly influence the expression of PAX6, SOX2 and OTX2 during neural induction. Conclusion FOXG1 gene large fragment knockout in hESCs can be quickly induced by a pair of gRNAs cotransfection. FOXG1 deletion has no significant impacts on neural induction of hESCs.