基于人类全基因组lncRNA的CRISPR筛选和RNA-seq数据的lncRNA特征分析

It is known that lncRNAs play important roles on regulation of gene expression, but the information of genomic features of whole-genome lncRNAs is very limited now. Our group studied large-scale lncRNAs by high-throughput genome editing (Nature biotechnology, 2016) and recently, our group developed a new method, which is the first achievement of knockout screening of human whole-genome lncRNAs, based on CRISPR technology. In the process of preparation for this project, I found two unreported phenomena: (1) a part of lncRNAs obviously regulate cell-growth but have very low expression levels; (2) the phenotype of cell growth is related with the GC content of lncRNA splicing sites. Therefore, by applying different machine learning methods such as MARS (Multivariate Adaptive Regression Splines) and principal component analysis, this project will analyze the unique data of functional screening of lncRNAs and total RNA-seq of 113 human tissues provided by the open database, combining with validation by biological experiments, to investigate the features of expression, genomic structure and function of lncRNAs. This project aims to provide biological clues of functional lncRNA features for systematic and comprehensive study of lncRNAs.

lncRNA在调控基因表达上起着重要作用，但在全基因组水平对lncRNA的特征的了解还十分有限。本课题组利用高通量基因组编辑手段对lncRNA的功能进行了大规模研究（Nature Biotechnology，2016），并在近期首次实现了人类全基因组范围的lncRNA的CRISPR敲除筛选（Nature Biotechnology审稿中），产生了大规模lncRNA功能性数据。申请人对相关数据进行了前期分析，发现部分极低表达的lncRNA能够显著调控细胞增殖；另外，lncRNA剪接位点的GC量与其表型也存在一定的相关性，鉴此，本项目将结合独有的全基因组lncRNA功能性数据和公共数据库中的113种人类组织的RNA-seq数据进行分析，通过主成分分析、MARS等机器学习方法，并结合湿实验，阐释lncRNA表达、基因结构与功能的特征及其关联，以期为系统和深入理解lncRNA的生物学特征提供依据。

lncRNA在细胞内有着多种多样的作用，但是当前对全基因组功能lncRNA的特征的了解还十分有限。本课题首先开发了基于CRISPR-Cas9系统的编码基因以及lncRNA功能元件检测的实验技术；然后使用本课题组近年发表的人类全基因组lncRNA的CRISPR敲除筛选数据，结合多种公开数据库提供的多维度功能性数据，探索了功能性lncRNA的基因组学特征。我们发现（1）lncRNA剪接位点的GC含量与lncRNA在人类不同组织器官中的表达水平上限、表达水平的特异性相关；（2）lncRNA在不同物种中的序列保守程度较低，但剪接位点GC含量高的lncRNA在不同物种间有较高的序列保守性；（3）具有重要功能的lncRNA有较高的细胞内源RNA A-to-I的单碱基位点的富集。本课题在lncRNA功能特征的新发现为探索更多的lncRNA功能提供了重要支撑。