Knocking Out KMT2D SET Domain In A Human Embryonic Stem Cell Line Using CRISPR/Cas9 System

Objective:Kabuki Syndrome?KS?is a rare genetic disease characterized by a unique facial appearance,cardiac abnormalities,skeletal abnormalities,immunodeficiency,and mild to moderate mental retardation.Studies have shown that55%-80%of KS cases are caused by KMT2D gene mutations.It is of great significance to study the pathogenesis of KMT2D-mediated KS.KMT2D is a H3K4methyltransferase,existing in many organs and tissues.The C-terminus of the protein contains a SET domain,which has methyltransferase activity and maintains the stability of the protein in the cell.To further study the regulatory role of KMT2D-mediated H3K4 methylation in neural development,this study constructed a KMT2D SET domain biallelic knockout in a human embryonic stem cell line using CRISPR/Cas9 technology.This cell line provides a reliable cell model to facilitate further investigation of disease mechanism.Methods:First,the genomic sequence of KMT2D was extracted to design sgRNAs.Multiple sgRNA pairs were designed at both sides of the SET domain.T7EI was used to detect their cutting efficiency and the sgRNA pair with the highest efficiency was selected and inserted into a pX330 vector.At the same time,we selected two 1 kb sequences flanking the cleavage site as homology arms.PCR specifically amplifies the homology arms and integrated Puro or Hygro resistance gene as a donor DNA..The two targeting sgRNAs and the two donor DNAs were transferred together into a human embryonic stem cell line HN4.After Puro and Hygro selection,single clones were selected and expanded in the culture as stable cell lines.Validation PCR,sequencing,and q-PCR identification methods were used to detect the expression of HN4 KMT2D SET Domain DNA and mRNA levels.Finally,the cell line was karyotyped to confirm whether the karyotype was correct.Results:The sgRNA plasmids targeting both ends of the KMT2D SET Domain were successfully constructed.The sgRNA pairs were tested for T7EI cleavage efficiency and the optimal sgRNA pair was selected.A donor was modified to contain KMT2D homology arms and a Puro or Hygro-resistant gene.Validation PCR results showed that 8/12 cells simultaneously incorporated both Puro and Hygro to replace the wild-type KMT2D,demonstrating the biallelic knockouts.The validating sequencing further confirmed that these cells underwent homologous recombination of the target fragment in both alleles.The q-PCR results showed that at the mRNA level,the SET Domain was no longer expressed,whereas the expression of other regions remained unaltered.The karyotype test proved that the KMT2D SET^-/-cell line had a normal karyotype.Conclusion:The pX330-sgRNA plasmid was successfully constructed and its efficiency was verified.A donor plasmid with either Puro or Hygro to replace the KMT2D SET Domain was successfully constructed.The karyotype test proved that the cell line had a normal karyotype.A stable KMT2D gene knockout human embryonic stem cell line was successfully established.This cell line provides an ideal experimental model to study the physiological function of the KMT2D gene and reveal the epigenetic mechanism of KS caused by KMT2D mutation.Moreover,this biallelic knockout strategy exemplifies as a technical reference.