Abstract：
Many long noncoding RNAs (lncRNAs) have been identified through siRNA-based screening as essential regulators of embryonic stem cell (ESC) pluripotency. However, the biological and molecular functions of most lncRNAs remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 lncRNAs previously reported to promote pluripotency in mouse ESCs. Unexpectedly, all of these lncRNAs were dispensable for pluripotency maintenance and proliferation in mouse ESCs when disrupted individually or in combination. Single-cell transcriptomic analysis also showed that the knockout of these lncRNAs has a minimal impact on pluripotency gene expression and cell identity. We further showed that several small hairpin RNAs (shRNAs) previously used to knock down lncRNAs caused the downregulation of pluripotency genes in the corresponding lncRNA-knockout ESCs, indicating that off-target effects likely responsible for the pluripotency defects caused by these shRNAs. Interestingly, linc1343-knockout and linc1343-knockdown ESCs failed to form cystic structures and exhibited high expression of pluripotency genes during embryoid body (EB) differentiation. By reintroducing RNA products generated from the linc1343 locus, we found that two snoRNAs, Snora73a and Snora73b, but not lncRNAs, could rescue pluripotency silencing defects during EB differentiation of linc1343 knockout ESCs. Our results suggest that the 8 previously annotated pluripotency-regulating lncRNAs have no overt functions in conventional ESC culture; however, we identified snoRNA products derived from an annotated lncRNA locus as essential regulators for silencing pluripotency genes.
摘要：
通过基于siRNA的筛选，许多长链非编码RNA（lncRNA）已被鉴定为胚胎干细胞（ESC）多能性的基本调节因子。然而,大多数lncRNAs的生物学和分子功能仍不清楚。这里,我们采用CRISPR/Cas9介导的基因敲除技术来探索8种lncRNAs的功能,这些lncRNAs是先前报道的在小鼠ESCs中促进多能性的。出乎意料的是,当单独或联合破坏时,所有这些lncRNA对于小鼠ESC的多能性维持和增殖是不必要的.单细胞转录组分析还显示这些lncRNA的敲除对多能性基因表达和细胞同一性的影响最小。我们进一步表明，以前用于敲低lncRNAs的几种小发夹RNA（shRNAs）导致相应的lncRNA敲除ESC中多能性基因的下调，表明脱靶效应可能是由这些shRNA引起的多能性缺陷的原因。有趣的是,linc1343敲除和linc1343敲除的ESC未能形成囊性结构，并在胚状体（EB）分化过程中表现出多能性基因的高表达。通过重新引入从linc1343基因座产生的RNA产物，我们发现了两个snoRNA，Snora73a和Snora73b,但不是lncRNAs，可以挽救linc1343敲除ESCsEB分化过程中的多能性沉默缺陷。我们的结果表明，8个先前注释的多能性调节lncRNAs在常规ESC培养中没有明显的功能；然而，我们确定了源自注释的lncRNA基因座的snoRNA产物是沉默多能性基因的必需调节因子。
