Construction Of GATA1s Knockout HPSCs Model And Its Effect On Hematopoietic Differentiation

Objective To establish and validate GATA1s and GATA1 knockout human pluripotent stem cells(hPSCs)models,and to explore the physiological function changes during hematopoietic differentiation in vitro.Methods Vector construction:The targeting plasmid containing recombinant arm-knockin segments-LoxP-hygromycin screening markerLoxP-re combinant arm and gRNA plasmid containing gRNA-Cas9 were constructed,respectively.The knockin fragment of GATA1s knockout was GATA1 exon II-VI sequence and BGH polyA sequence.The knockin fragment in GATA1 knockout was the self-cleaving polypeptide P2A sequence.Target sequence knockin:the above target plasmid and corresponding gRNA plasmid were simultaneously introduced into hPSCs H1 cell line by electroporation.Clone screening and identification:After preliminary screening with hygromycin,the positive clones were further subjected to electroporation with Cre to recombine LoxP site specifically to remove screening markers,and further cultured by single cell cloning.PCR and product sequencing were used to identify the correct geneedited cells,and finally the expression of GATA1 and GATA1s proteins was verified by western blot(WB).The verified cell lines were induced to hematopoietic differentiation in vitro,and then analyzed by flow cytometry(FCM),Blood cell colony formation assay,single-cell transcriptome analysis,and chromatin immunoprecipitation assay(ChIP).Overexpression of.GATA1 in GATA1s knockout cell lines was further analyzed by FCM and blood cell colony culture.Results The monoclonal cell lines after electroporation with targeting plasmid,gRNA plasmid and Cre expression plasmid were identified by PCR with respective primers.Finally,3 strains were screened out with GATA1s knockout and 3 strains were screened out with GATA1 knockout,all of which met the expected band.The final WB results showed that the wild type had GATA1 and GATA1s bands,while the GATA1s knockout cell line only had GATA1 bands,and the GATA1 knockout cell line only had GATA1s bands.The GATA1s knockout and GATA1 knockout H1 cell lines were successfully constructed.FCM analysis showed that the proportion of CD34+CD43+ and CD34+CD45+cells increased in GATA1s or GATA1 knockout group on day 12 of differentiation,but the proportion of GPA+ cells decreased significantly.Further induced megakaryocytic and erythroid differentiation.It was found that CD41a+CD42b+cells and CD71+GPA+cells were significantly reduced or even absent.Colony culture of blood cells also lacked erythroid-associated colonies.Moreover,addition of GATA1 to the GATA1s KO cell line failed to rescue the erythroid and megakaryocyte deletion phenotypes.Single-cell transcriptome sequencing analysis combined with ChIP analysis showed that GATA1s knockout reduced hematopoietic stem cells(HSCs),erythrocytes and megakaryocytes,and increased mast and NK cells.In addition to GATA1s binding to HSCs production and erythroid megakaryocyte development,we found that GATA1s knockout affected the expression of GATA2,PU.1,HIF1α.Conclusion GATA1s plays an important role in the maintenance of HSCs during normal hematopoietic differentiation,and plays an indispensable role in the differentiation of erythroid and megakaryocyte lineages.