Establishment Of MSX1 Gene Knockout Human Embryonic Stem Cell Lines By CRISPR-Cas9 Technology

Objectives:1.To construct a congenital tooth agenesis related gene MSX1 knockout human embryonic stem cell(hESc)lines by CRISPR-Cas9 gene editing technology.2.To study the pluripotency of MSX1 gene konckout hESc line.3.To investigate the ability to differentiate into outer,middle,and mesodermal cells of MSX1 gene konckout hESc line embryoid body.Methods:1.pX330-MSX1KO-sgRNA vector and MSX1 gene knockout homologous recombinant vector(donor)were constructed by molecular cloning technique.2.pX330-MSX1KO-sgRNA vector and donor were electriciticted into hESc,puromycin resistant colonies were picked by PCR detection and sequencing identification.3.The pluripotency detection of MSX1 homozygous konckout hESc(H1-MSX1-DKO)were performed by morphologic observation,karyotype analysis,flow cytometry.4.MSX1 homozygous konckout hESc embryond body were formed and induced differentiated into outer,middle,and mesodermal cells in virto.Total RNA of D0 and D8 cells were extracted and real-time qPCR reaction was performed,then the expression of three embryonic layer cell markers were detected.Results:1.Sanger sequencing results showed that pX330-MSX1KO-sgRNA vector and donor were constructed successfully.78 heterozygous and 2 homozygous knockout cell colonies(H1-MSX1-SKO,H1-MSX1-DKO)were obtained.2.These cells exhibited classical human embryonic stem cell morphology and had normal karyotype.Flow cytometry analysis showed high expression of pluripotent cell markers OCT4 and SSEA4 of H1-MSX1-DKO.3.qPCR results showed that the three embryonic layer cell markers of H1-MSX1-DKO EB cells in D8 were higher than the cells in D0 in different degree.Conclusion:Our results supported the application of CRISPR-Cas9 gene editing technique in human embryonic stem cells.MSX1 gene knockout hESc maintained the pluripotency of the stem cell and retained the ability to differentiate into three germ layers in vitro.Furthermore,the MSX1 knockout human embryonic stem cell lines would provide good materials for the further studies of tooth development molecular mechanism and then provide reference for our treatment of congenital tooth agenesis.