Heterozygous and homozygous knock-in of PIK3CA-H1047R into human iPSCs

We used CRISPR/Cas9 to knock in the cancer "hotspot" mutation PIK3CA-H1047R into one or both alleles of a wild-type induced pluripotent stem cell (iPSC) line (WTC11; Coriell # GM25256; P37-P38). Three clones from each genotype (wild-type, heterozygous, homozygous) were subjected to single-end mRNA sequencing (mean read depth per sample: 20 million) to determine whether PIK3CA-H1047R exerts allele dose-dependent transcriptional effects. Multidimensional scaling demonstrated distinct transcriptomic signatures of wild-type, heterozygous and homozygous cells. The transcriptome of heterozygous cells was nearly identical to wild-type controls, with only 131 differentially-expressed transcripts (FDR = 0.05). In contrast, homozygosity for PIK3CA-H1047R led to differential expression of 1,914 genes. This indicates widespread transcriptional remodeling with a sharp allele dose-dependency, suggestive of a threshold effect.

本研究利用CRISPR/Cas9系统，将癌症“热点”突变PIK3CA-H1047R敲入野生型诱导多能干细胞（induced pluripotent stem cell, iPSC）系WTC11（Coriell数据库编号GM25256，传代37至38代）的一个或两个等位基因。针对每种基因型（野生型、杂合型、纯合型）各选取3个克隆，开展单端mRNA测序（single-end mRNA sequencing），每个样本的平均测序深度为2000万条reads，以此探究PIK3CA-H1047R是否存在等位基因剂量依赖性的转录调控效应。多维尺度分析（Multidimensional scaling）结果显示，野生型、杂合型与纯合型细胞具备截然不同的转录组特征。杂合型细胞的转录组与野生型对照组几乎完全一致，仅存在131个差异表达转录本（错误发现率（False Discovery Rate, FDR）=0.05）。与之相反，PIK3CA-H1047R纯合突变可导致1914个基因发生差异表达。上述结果表明，该突变引发了广泛的转录组重编程，且呈现显著的等位基因剂量依赖性，提示存在阈值效应。