利用CRISPR/Cas9技术构建KMT2D基因敲除的人胚胎干细胞系

Knocking out KMT2D in a human embryonic stem cell line using CRISPR/Cas9 system

[目的]通过CRISPR/Cas9技术构建KMT2D SET结构域双等位基因敲除的人胚胎干细胞HN4细胞系。[方法]通过CRISPR在线设计工具在SET结构域两侧各设计多条sgRNA,利用T7EI选出效率最高的sgRNA,构建到pX330载体中,特异性扩增同源臂,分别构建到双抗性载体中作为Donor。将sgRNA与Donor共同电转至HN4细胞中,经过Puro和Hygro抗性筛选后,挑取单克隆,利用PCR,测序以及q-PCR鉴定等方法鉴定细胞的基因型。[结果]成功构建了靶向KMT2D SET Domain两端的sgRNA的表达质粒,并选择出切割效率最高的sgRNA。PCR鉴定结果显示,有8株细胞可同时扩增出Puro和Hygro两个目的片段,且无野生型条带。通过测序和序列比对确认KMT2D SET已被敲除,双等位基因分别被Puro和Hygro替换。q-PCR结果显示,mRNA层面上,SET Domain已经不表达,而其他位点则不受影响。[结论]成功构建pX330-sgRNA质粒,并验证其具有活性。成功构建了靶向KMT2D SET Domain的分别含有Puro和Hygro不同抗性的Donor。成功建立了稳定的KMT2D基因敲除的人胚胎干细胞系。

[Objective]Using CRISPR/Cas9 to knock-out KMT2D SET domain in a human embryonic stem cell line.[Method]Multiple sgRNAs were designed on both sides of the SET domain using the CRISPR online design tool.The cleavage efficiency was evaluated by T7 endonuclease digestion,and the most efficient sgRNAs at the 5'and 3'of the SET domain were ligated into the pX330 vector as the targeting plasmids.Meanwhile,1 kb 5'and 3'arms located at the upstream and downstream of the targeted knock-out sequence,correspondingly,were selected for homologous recombination.Subsequently,5'and 3'homology arms were integrated into a donor vector containing Puromycin(Puro)or Hygromycin B(Hygro)-resistance gene.These two donor vectors,together with two targeting plasmids,were co-transfected into the HN4 hESC cell line.After screened for Puro and Hygro double resistance,single clones were selected and expanded.Finally,PCR,second-generation sequencing,and qPCR were applied to validate the genotype of the selected clones.[Result]The expression plasmids targeting the sgRNA at both ends of KMT2D SET Domain had been successfully constructed,and the sgRNA with the highest cutting efficiency had been selected.The results of PCR insertion identification and single-double knock identification showed that 8 cells could simultaneously amplify two target fragments of Puro and Hygro,and these cells had no wild-type fragments.Sequencing identification and sequence comparison confirmed that KMT2D SET has been knocked out with no base mutation,and the biallelic genes were replaced by Puro and Hygro,respectively.Q-PCR showed that the SET Domain was not expressed,while other sites were not affected at the mRNA level.[Conclusion]The pX330-sgRNA plasmid was successfully constructed and verified to be active.Successfully constructed Donor targeting KMT2D SET Domain containing Puro and Hygro with different resistance.These results verified that the KMT2D SET domain KO cell line was successfully constructed.