Genomic deletion by CRISPR/Cas9 reveals a critical role for Evx1as/Evx1 locus in regulating embryonic stem cell differentiation. Mus musculus

We report that head to head long noncoding RNAs contribute to transcription and developmental process. Thousands of lncRNAs have been identified in the whole genome, and classified to several subgroups based on the genome position with their coding neighbors. XH, the head to head subgroup is associated with transcription and development in GO analysis. Here, we deleted the promoter and exon1 region which shared by Evx1as/Evx1 XH pair in embryonic stem cells by two sgRNAs-mediated CRISPR. Two homozygous mutants came out from 46 single clones with high efficiency, while two control clones were picked from cells which were only Cas9 transfected. These two mutants failed to activate Evx1 in -LIF differentiation compared to control cells, while Evx1as still have 30% mRNA residual. Meanwhile, we characterized gene expression level among different stages of ESC differentiation and found many differentiated related makers exhibited aberrant regulation, especially endodermal genes. We propose that XH lncRNA and its coding neighbor could regulate each other, and function as a unit to promote ESC differentiation and biological development. Overall design: All RNA-seq(s) were designed to reveal the differentially expressed genes between wild-type or Evx1as/Evx1 promoter and exon1 KO cells during -LIF differentiation.

本研究报道，头对头型长链非编码RNA（long noncoding RNA，lncRNA）参与转录调控与发育过程。全基因组范围内已鉴定出数千条长链非编码RNA，并可根据其与编码基因邻接的基因组位置划分为多个亚型。其中XH亚组（属于头对头型lncRNA）经基因本体（Gene Ontology，GO）分析显示与转录及发育过程密切相关。
本研究通过两条单引导RNA（single guide RNA，sgRNA）介导的CRISPR基因编辑系统，在胚胎干细胞（embryonic stem cell，ESC）中敲除了Evx1as/Evx1头对头型XH亚组共有的启动子及外显子1区域。从46个单克隆细胞株中高效获得2株纯合突变体，同时仅转染Cas9的细胞中筛选得到2株对照克隆。
在不含白血病抑制因子（leukemia inhibitory factor，LIF）的分化体系中，与对照细胞相比，两株突变体均无法激活Evx1的表达，而Evx1as仍残留30%的mRNA水平。同时，本研究对胚胎干细胞分化不同阶段的基因表达水平进行了表征，发现众多分化相关标志物呈现异常调控，以内胚层基因尤为显著。
本研究提出，XH型长链非编码RNA与其相邻的编码基因可相互调控，并以功能单元的形式促进胚胎干细胞分化与生物发育过程。
实验整体设计：所有RNA测序（RNA sequencing，RNA-seq）实验均旨在揭示不含白血病抑制因子的分化体系中，野生型细胞与Evx1as/Evx1启动子及外显子1敲除细胞之间的差异表达基因。