The CRISPR/Cas9 technology was used to achieve the H2-K1 gene knockout of mouse ES cells

which will be useful for generation of MHC class Ⅰ gene humanized mice. In this study

two sgRNAs were designed

which are targeting to the Exon2 and Exon3 of H2-K1

respectively. The plasmids expressing the sgRNAs were constructed using pX330 as the matrix plasmid. In order to knockout H2-K1

the constructed plasmids and pSUPERpuro were cotransfected into the mES cells through electroporation. After screening by puromycin

the result of gene targeting was determined by PCR，and H2-K1 knockout mouse ES cells were further confirmed through sequencing and flow cytometry. We found that H2-K1 in the mouse ES cell was knocked out using CRISPR/Cas9 technology. Through PCR

4 clones were determined as one allele knockout(19.0%)

2 clones were determined as two allele knockout(9.5%). 2 clones were further confirmed as two allele knockout clones by sequencing and flow cytometry. The generated H2-K1 knockout mouse ES cells would provide a reference for the knockout and replacement of MHC class Ⅰ gene.