不同打靶位点影响人诱导多能干细胞MYO7A杂合突变位点的基因校正中CRISPR/Cas9介导的同源重组效率

应用CRISPR-Cas9系统对人诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）进行基因编辑，为疾病模型的建立、致病机制研究、药物筛选及基因校正治疗疾病提供了更广阔的平台。相对于CRISPR-Cas9介导的基因敲除，应用该系统介导的同源重组实现基因点突变或突变校正效率要低、且难度偏大。为了实现对MYO7A杂合点突变（c.4118C>T）的人iPSCs的点突变校正，本文构建了表达maxGFP的pX330质粒。针对需校正的突变位点，设计5组识别序列并连接到maxGFP-pX330中构建靶向质粒。将5组打靶质粒分别转染HEK 293FT细胞48 h，细胞表达GFP；测序结果显示，MYO7A基因相应位点出现杂峰，表明打靶质粒具有打断活性。将同源模版单链寡核苷酸链（single-stranded DNA oligonucleotides, ssODN）与打靶质粒共同电转入人iPSCs后48 h，经流式分选出(5.8±2.2)%的细胞表达GFP。分选后细胞行单克隆扩增并测序。结果显示，打靶质粒1和ssODN组合对点突变校正未成功；打靶质粒2、3、4、5与ssODN组合均获得了校正后的细胞株。本研究表明，打断位点是影响同源重组校正效率的关键因素。当应用CRISPR/Cas9（或其它核酸酶）介导的同源重组进行基因编辑操作时，可以同时选择多个打靶位点造成基因组不同位置上的双链打断（double-stranded break, DSB）位点，以获得目的单克隆细胞株。本研究为应用CRISPR-Cas9系统对人诱导多能干细胞进行基因编辑提供了有力参考。

CRISPR/Cas9-mediated Homologous Recombination in Human iPSCs to Correct MYO7A Heterozygous Point Mutation Is Determined byDouble-strand Break Targeting Sites

CRISPR-Cas9-mediated precise genomic editing in human induced pluripotent stem cells (hiPSCs) is a powerful technology for disease modeling, study of pathological mechanism，drug screening and future patient-specific cellular therapies. The efficiency of gene correction of the point mutation using CRISPR-Cas9-mediated homologous recombination (HR) is much lower than that of gene knock-out made by CRISPR-Cas9-mediated nonhomologous end joining (NHEJ). In this study, we intended to correct the heterozygous point mutation c.4118C>T of MYO7A in human iPSCs by CRISPR-Cas9-mediated HR. Firstly，the plasmid pX330 expressing GFP was constructed and five guide sequences targeting five different genome sites were designed and linked into the plasmid separately. Then the maxGFP-expressing guideRNA-Cas9 plasmids were transfected into HEK 293FT cells to evaluate their cleavage activities. After transfection for 48 h, the sequencing results displayed scrambled peaks around the targeting sites, which indicated the effective cleavage activities of all the five maxGFP-expressing guideRNA-Cas9 plasmids. The maxGFP-expressing guideRNA-Cas9 plasmids and ssODN (single-stranded DNA oligonucleotides) were transfected into human iPSCs. After transfection for 48 h, 5.8%±2.2% cells expressing GFP were sorted and cultured to form cell clones. Gene sequencing of MYO7A around mutation sites in these cell clones indicated that the heterozygous point mutation was homozygously corrected among the cells transfected with guideRNA2/3/4/5-Cas9 plasmids and ssODN, but not in cells transfected with guideRNA1-Cas9 plasmids and ssODN. This study indicates that it is the double-stranded break targeting site, to some extent, that determines the gene correction efficiency conducted by CRISPR/Cas9-mediated HR. Researchers should design several guide sequences to target different sites of the gene when they intend to make gene editing successful through CRISPR-Cas9 (or any other nuclease)-mediated HR. This study offers an effective reference for gene editing of human iPSCs through CRISPR-Cas9 mediated HR.