Zhang Hanwen, Zhang Siwei
Center for Psychiatric Genetics, Research Institute, NorthShore University HealthSystem, Evanston, IL 60201, USA.
Bio Protoc. 2021 Jun 20;11(12):e4051. doi: 10.21769/BioProtoc.4051.
Human induced pluripotent stem cells (hiPSCs) have been extensively used in the fields of developmental biology and disease modeling. CRISPR/Cas9 gene editing in iPSC lines often has a low frequency, which hampers its application in precise allele editing of disease-associated single nucleotide polymorphisms (SNPs), especially those in the noncoding parts of the genome. Here, we present a unique workflow to engineer isogenic iPSC lines by SNP editing from heterozygous to homozygous for disease risk alleles or non-risk alleles using a transient and straightforward transfection-based protocol. This protocol enables us to simultaneously obtain pure and clonal isogenic lines of all three possible genotypes of a SNP site within about 4 to 5 weeks.
人类诱导多能干细胞（hiPSC）已广泛应用于发育生物学和疾病建模领域。iPSC系中的CRISPR/Cas9基因编辑频率往往较低，这阻碍了其在疾病相关单核苷酸多态性（SNP）精确等位基因编辑中的应用，尤其是那些位于基因组非编码区的SNP。在这里，我们展示了一种独特的工作流程，通过基于瞬时和直接转染的方案，将疾病风险等位基因或非风险等位基因的单核苷酸多态性从杂合子编辑为纯合子，从而构建同基因iPSC系。该方案使我们能够在大约4至5周内同时获得一个SNP位点所有三种可能基因型的纯合和克隆同基因系。
