2019.01.08，单革教授实验室发表文章，报导了其实验室最新的研究成果。单革实验室在2017年发表的Developmental Cell文章基础上，于Genome Biology发表了题为《Systematic evaluation of C. elegans lincRNAs with CRISPR knockout mutants》的文章，报导通过优化的CRISPR-cas9 系统对秀丽线虫中155个基因间的长链非编码RNA(lincRNA)进行逐一敲除（秀丽线虫已知的全部lincRNA共170个），系统地研究了秀丽线虫中lincRNA的功能。这也是第一篇在多细胞动物中利用CRISPR-cas9技术、在全基因组水平上、对一种特定类型长非编码RNA进行敲除并系统分析其生理功能及功能机理的研究。对155个lincRNA敲除突变体进行六个方面表型的筛选发现23个lincRNA突变体分别在其中一个或者两个生理表型上具有不同程度的缺陷。通过对秀丽线虫不同发育时期的转录组测序进行分析，作者研究了lincRNA 和mRNA共表达情况，同时建立了lincRNA和microRNA共表达及调控网络。通过对秀丽线虫不同发育时期近300个转录因子的ChIP-seq分析来探究转录因子在线虫不同发育阶段对lincRNA的调控，进而研究了这23个lincRNA行使其生理调控功能的机理。本研究系统地探索了lincRNA在多细胞动物中的生理功能，一定程度拓宽了lincRNA研究领域，同时该研究获得的lincRNA敲除线虫株为后续进一步研究长链非编码RNA在衰老和疾病等中的作用提供了有力支持。文章的共同第一作者是博士生卫帅、博士后陈禾、以及国际学生Emmanuel Enoch Dzakah（最近已博士毕业）。
该研究得到了科技部、国家基金委、以及中科院“衰老的生物学基础和干预策略”先导科技专项（培育）的经费支持。
Shuai Wei*, He Chen*, Emmanuel Enoch Dzakah*, Bin Yu, Xiaolin Wang, Tao Fu, Jingxin Li, Lei Liu, Shucheng Fang, Weihong Liu, Ge Shan. Systematic evaluation of C. elegans lincRNAs with CRISPR knockout mutants.Genome Biology, 2019; 20:7.
论文链接：https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1619-6
Abstract
Long intergenic RNAs (lincRNAs) play critical roles in eukaryotic cells, but systematic analyses of the lincRNAs of an animal for phenotypes are lacking. We generate CRISPR knockout strains for Caenorhabditis elegans lincRNAs and evaluate their phenotypes.
C. elegans lincRNAs demonstrate global features such as shorter length and fewer exons than mRNAs. For the systematic evaluation of C. elegans lincRNAs, we produce CRISPR knockout strains for 155 of the total 170 C. elegans lincRNAs. Mutants of 23 lincRNAs show phenotypes in 6 analyzed traits. We investigate these lincRNAs by phenotype for their gene expression patterns and potential functional mechanisms. Some C. elegans lincRNAs play cis roles to modulate the expression of their neighboring genes, and several lincRNAs play trans roles as ceRNAs against microRNAs. We also examine the regulation of lincRNA expression by transcription factors, and we dissect the pathway by which two transcription factors, UNC-30 and UNC-55, together control the expression of linc-73. Furthermore, linc-73 possesses a cis function to modulate the expression of its neighboring kinesin gene unc-104 and thus plays roles in C. elegans locomotion.
By using CRISPR/cas9 technology, we generate knockout strains of 155 C. elegans lincRNAs as valuable resources for studies in noncoding RNAs, and we provide biological insights for 23 lincRNAs with the phenotypes identified in this study.