a Department of Biochemistry , University of Washington , Seattle , Washington 98195 , USA.
b Institute for Stem Cell and Regenerative Medicine , University of Washington , Seattle , Washington 98109 , USA.
Cell Cycle. 2018;17(5):535-549. doi: 10.1080/15384101.2018.1442621. Epub 2018 Apr 5.
To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein.
为了方便编辑原始态人类胚胎干细胞（hESC）的基因组，我们将一个可诱导 Cas9 的双盒式元件引入原始态 hESC 的 AAVS1 位点（iCas9）。iCas9 系保留了核型稳定性、多能性标记物的表达、分化潜能以及在 5iLA 和 EPS 多潜能条件下的稳定性。该 iCas9 系通过 CRISPR-Cas9 系统诱导高效同源定向修复（HDR）和非同源末端连接（NHEJ）突变。我们利用 iCas9 系来研究早期人类多潜能性中的表观遗传调节因子 PRC2。PRC2 的需求区分了早期多潜能性阶段，然而，在这些阶段中调节 PRC2 活性的因素尚不清楚。我们发现 JARID2 2iL-I-F hESC 突变体中的 H3K27me3 和多能性标记物减少，表明 JARID2 在维持 hESC 2iL-I-F 状态中是必需的。这些数据表明 JARID2 在 2iL-I-F 状态下调节 PRC2，而在 5iLA 状态下缺乏 PRC2 功能可能是由于缺乏足够的 JARID2 蛋白。
