Genetics of long non-coding RNAs in zebrafish

描述(申请人提供)：拟议研究的长期目标是确定长非编码RNA在脊椎动物发育中的功能。在过去的十年里，全基因组研究出现了爆炸式的增长，发现了太多以前没有注释的转录本。在这些新的转录本中，一类长的非编码RNA(LncRNAs)引起了特别的关注，因为它们代表了基因组中一个广泛的、基本上未被探索的组成部分：基因组的“暗物质”。这项拟议的研究旨在进行反向遗传筛选，并分析斑马鱼中100个lncRNAs的功能。基因组学和计算方法导致了在斑马鱼发育过程中表达的数百个lncRNAs的鉴定。根据保守性、基因组位置、表达和染色质图谱选择的100个这些lncRNAs将被破坏。反义寡核苷酸将被用来直接干扰lncRNA，而位点特异性内切酶(TALEN和CRISPR)将被用来干扰lncRNA基因。然后，胚胎和幼虫将进行系统的表型鉴定，以揭示lncRNAs的功能。拟议的反向遗传筛查将提供第一个全面分析脊椎动物在体内的lncRNA功能，并为该领域提供资源，以揭示这一神秘类别的分子的作用。

DESCRIPTION (provided by applicant): The long-range goal of the proposed studies is to determine the functions of long non-coding RNAs in vertebrate development. The past decade has seen an explosion of genome-wide studies that have identified a plethora of previously unannotated transcripts. Among these novel transcripts, the class of long non-coding RNAs (lncRNAs) has attracted particular attention since they represent an extensive, largely unexplored component of the genome: the "dark matter" of the genome. The proposed study aims to perform a reverse genetic screen and analyze the function of 100 lncRNAs in zebrafish. Genomics and computational approaches have led to the identification of several hundred lncRNAs expressed during zebrafish development. 100 of these lncRNAs, selected by conservation, genomic location, expression, and chromatin profile, will be disrupted. Antisense oligonucleotides will be used to directly interfere with lncRNAs, and site-specific endonucleases (TALENs and CRISPRs) will be employed to disrupt lncRNA genes. Embryos and larvae will then undergo systematic phenotypic characterization to reveal the functions of lncRNAs. The proposed reverse genetic screen will provide the first comprehensive analysis of vertebrate lncRNA function in vivo and generate the resources for the field to reveal the roles of this mysterious class of molecules.