Joung Julia, Engreitz Jesse M, Konermann Silvana, Abudayyeh Omar O, Verdine Vanessa K, Aguet Francois, Gootenberg Jonathan S, Sanjana Neville E, Wright Jason B, Fulco Charles P, Tseng Yuen-Yi, Yoon Charles H, Boehm Jesse S, Lander Eric S, Zhang Feng
Department of Biological Engineering, MIT, Cambridge, Massachusetts 02139, USA.
Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.
Nature. 2017 Aug 17;548(7667):343-346. doi: 10.1038/nature23451. Epub 2017 Aug 11.
Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes-for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.
哺乳动物基因组包含数千个转录长链非编码RNA（lncRNA）的基因座，其中一些已知通过多种机制在不同的细胞过程中发挥关键作用。尽管一些lncRNA基因座编码的RNA可在非局部发挥作用（反式作用），但越来越多的证据表明，许多lncRNA基因座在局部发挥作用（顺式作用），以调节附近基因的表达——例如，通过lncRNA启动子、转录或转录本本身的功能。尽管它们可能具有重要作用，但识别功能性lncRNA基因座并区分这些机制与其他机制仍然具有挑战性。在这里，为应对这些挑战，我们开发了一种全基因组规模的CRISPR-Cas9激活筛选方法，该方法针对10000多个lncRNA转录起始位点，以识别影响感兴趣表型的非编码基因座。我们发现11个lncRNA基因座，在招募激活剂后，可介导人黑色素瘤细胞对BRAF抑制剂的抗性。大多数候选基因座似乎调节附近的基因。对一个名为EMICERI的候选基因座的详细分析表明，其转录激活导致四个相邻蛋白质编码基因的剂量依赖性激活，其中一个基因赋予抗性表型。我们的筛选和表征方法提供了一个CRISPR工具包，可用于系统地发现非编码基因座的功能，并阐明它们在基因调控和细胞功能中的不同作用。
