Fogarty Norah M E, McCarthy Afshan, Snijders Kirsten E, Powell Benjamin E, Kubikova Nada, Blakeley Paul, Lea Rebecca, Elder Kay, Wamaitha Sissy E, Kim Daesik, Maciulyte Valdone, Kleinjung Jens, Kim Jin-Soo, Wells Dagan, Vallier Ludovic, Bertero Alessandro, Turner James M A, Niakan Kathy K
Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
NIHR Cambridge Biomedical Research Centre hIPSC Core Facility, Department of Surgery, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0SZ, UK.
Nature. 2017 Oct 5;550(7674):67-73. doi: 10.1038/nature24033. Epub 2017 Sep 20.
Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.
尽管其具有基本的生物学和临床重要性，但调节人类胚胎中首次细胞命运决定的分子机制仍未得到充分了解。在此，我们使用CRISPR-Cas9介导的基因组编辑来研究多能性转录因子OCT4在人类胚胎发育过程中的功能。我们使用基于诱导性人类胚胎干细胞的系统和小鼠受精卵显微注射，鉴定出一种高效靶向OCT4的引导RNA。使用这些优化方法，我们在二倍体人类受精卵中高效且特异性地靶向编码OCT4（POU5F1）的基因，发现囊胚发育受到损害。转录组学分析显示，在缺乏POU5F1的细胞中，不仅胚外滋养外胚层基因（如CDX2）的基因表达下调，而且包括NANOG在内的多能性上胚层调节因子的基因表达也下调。相比之下，缺乏Pou5f1的小鼠胚胎维持了直系同源基因的表达，囊胚发育得以建立，但维持过程受到损害。我们得出结论，CRISPR-Cas9介导的基因组编辑是在人类发育背景下研究基因功能的有力方法。
