Genome-wide expression analysis of yeast with CRISPR-mediated inhibition of GAL10 ncRNA compared to wild-type.

We analyzed the genome-wide expression by RNA-seq of a yeast strain that expresses Cas9d and a guideRNA targeted to the GAL10 locus (called +116), which inhibits GAL10 ncRNA expression from the antisense strand. We compared this strain to a strain expressing a scrambled guideRNA. The goal was to examine the effects of ncRNA inhibition and to examine if CRISPR inhibition of gene expression has off-target effects. We find that CRISPR-mediated inhibtion of GAL10 ncRNA only significantly changes expression of transcripts at the GAL1-10 locus, showing that CRISPR is highly specific, and that GAL10 ncRNA only control genes at the GAL locus. RNA-seq of 2 strains with CRISPR scrambled and 2 strains with CRISPR +116, the latter of which inhibits GAL10 ncRNA

我们通过RNA测序（RNA-seq）分析了表达Cas9d以及靶向GAL10基因座的向导RNA（guideRNA，命名为+116）的酵母菌株的全基因组表达谱；该向导RNA可抑制反义链上GAL10非编码RNA（ncRNA）的转录。我们将该菌株与表达随机序列向导RNA的对照菌株进行了比对。本研究旨在探究非编码RNA抑制的生物学效应，同时验证成簇规律间隔短回文重复序列（CRISPR）介导的基因表达抑制是否存在脱靶效应。我们发现，CRISPR介导的GAL10非编码RNA抑制仅会显著改变GAL1-10基因座处的转录本表达水平，这表明CRISPR介导的抑制具有高度特异性，且GAL10非编码RNA仅调控GAL基因座处的基因。本研究共对2株携带CRISPR随机向导RNA的菌株与2株携带CRISPR +116的菌株进行了RNA测序，其中后者可抑制GAL10非编码RNA的表达。