A CRISPR/Cas13 approach for identifying individual transcript isoform function in cancer

用于识别癌症中个体转录亚型功能的 CRISPR/Cas13 方法

基本信息

批准号：
10671680
负责人：
David Arthur Knowles
金额：
$ 34.61万
依托单位：
NEW YORK GENOME CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10671680
关键词：
Address Affect Alternative Splicing Binding Biological Assay Cancer cell line Cell Proliferation Cell physiology Cells Clustered Regularly Interspaced Short Palindromic Repeats Code Computer Analysis Data Disease Drops Drug resistance Essential Genes Exons Genes Grant Guide RNA Health Human Individual Libraries Link Machine Learning Malignant Neoplasms Mammalian Cell Measures Mediating Messenger RNA Methodology Methods Molecular Neoplasm Metastasis Performance Phenotype Production Proliferating Protein Isoforms Proteins Publishing RNA RNA Binding RNA Interference RNA Splicing Ribonucleases Role Structure System Testing Transcript Tumor Cell Line Work analytical method cancer therapy cell type computational pipelines computerized tools design genome-wide in vivo knock-down mRNA Precursor novel promoter therapeutic RNA tool transcriptome transcriptome sequencing transcriptomics tumor tumorigenesis web site

项目摘要

Abstract One of the major challenges in characterizing transcript isoform cellular function in health and disease is the lack of methods to specifically and efficiently downregulate their expression. RNA-targeting RNA-guided type VI CRISPR/Cas13 systems constitute a recently developed tool to knockdown transcripts in mammalian cells. Their RNase activity is activated by binding of a CRISPR RNA guide (gRNA) complementary to a single-stranded RNA target. The CRISPR/Cas13 systems tested to date have been shown to achieve highly specific knockdown of endogenous transcripts, with minimal off-target effects, outperforming current methodologies such as RNAi. While CRISPR/Cas13 has been shown to work efficiently when targeted to the pre-mRNA, transcriptome-wide strategies that would allow knockdown of transcript isoforms by targeting unique junctions in the mature mRNA molecule have not been explored. Selecting the best gRNA sequences is crucial for successful and specific RNA knockdown mediated by Cas13. Preliminary data from our lab shows that using gRNAs targeting sequences spanning exon-exon junctions in mature mRNA molecules with CRISPR/Cas13d (the smallest Cas13 effector) efficiently reduces transcript expression up to 80%. Moreover, targeting isoform-specific junctions allows for their individual knockdown without affecting non-targeted isoforms. These results suggest that there is no steric constraint in targeting junctions in mature mRNA molecules. Transcriptome analyses of tumors and cancer cell lines have comprehensively described the expression levels and identities of alternative transcript isoforms. In parallel, a number of studies have shown that splicing dysregulation is a hallmark of cancer. The causal link between transcript isoform expression and cancer related phenotypes has not been thoroughly studied. Here, we will expand the applicability of CRISPR/Cas13 systems to the systematic study of transcript isoforms in cancer by 1) combining large-scale experimental data with machine learning approaches to define rules for gRNA design when targeting specific transcript junctions 2) optimizing a pipeline and the computational analysis required for using CRISPR/Cas13 systems in forward transcriptomic pooled screens to interrogate the cellular function of transcript isoforms. By broadening the application of the CRISPR/Cas13 system, our pipeline will provide the molecular tools and computational analysis required for interrogating transcript isoform function in a robust, unbiased and highly expandable manner. We expect that our CRISPR/Cas13 approach will be able to overcome the limitations of current methods in identifying cell-specific isoform expression and/or ratios underlying tumorigenesis and drug resistance. Lastly, the CRISPR/Cas13 approach could be further adapted to perform targeting of transcript isoforms in vivo as an RNA-based therapeutic.

抽象的表征健康和疾病中转录亚型细胞功能的主要挑战之一是缺乏特异性和有效地下调其表达的方法。 RNA 靶向 RNA 引导的 VI 型 CRISPR/Cas13 系统是最近开发的一种工具，用于敲低哺乳动物细胞中的转录本。他们的 RNase 活性通过与单链 RNA 互补的 CRISPR RNA 向导 (gRNA) 的结合来激活目标。迄今为止测试的 CRISPR/Cas13 系统已被证明可以实现高度特异性的敲低内源转录本具有最小的脱靶效应，优于当前的方法（例如 RNAi）。虽然 CRISPR/Cas13 已被证明在靶向前 mRNA 时可以有效工作，但在转录组范围内通过靶向成熟 mRNA 中的独特连接点来敲除转录亚型的策略分子尚未被探索。选择最佳 gRNA 序列对于成功和特异性 RNA 至关重要由 Cas13 介导的敲低。我们实验室的初步数据表明，使用 gRNA 靶向序列使用 CRISPR/Cas13d（最小的 Cas13 效应子）跨越成熟 mRNA 分子中的外显子-外显子连接有效降低转录表达高达 80%。此外，针对同种型特异性连接点允许它们个体敲低而不影响非目标亚型。这些结果表明不存在空间位阻成熟 mRNA 分子中靶向连接的限制。肿瘤和癌细胞的转录组分析线全面描述了替代转录亚型的表达水平和身份。在与此同时，许多研究表明剪接失调是癌症的一个标志。因果关系转录亚型表达与癌症相关表型之间的关系尚未得到彻底研究。这里，我们将扩展 CRISPR/Cas13 系统的适用性，以系统研究转录亚型 1）将大规模实验数据与机器学习方法相结合来定义癌症的规则针对特定转录物连接点的 gRNA 设计 2) 优化流程和计算分析在正向转录组集合筛选中使用 CRISPR/Cas13 系统来询问细胞所需的转录亚型的功能。通过扩大 CRISPR/Cas13 系统的应用，我们的产品线将提供询问转录亚型功能所需的分子工具和计算分析稳健、公正且高度可扩展的方式。我们期望我们的 CRISPR/Cas13 方法能够克服当前方法在识别细胞特异性亚型表达和/或比率方面的局限性潜在的肿瘤发生和耐药性。最后，CRISPR/Cas13 方法可以进一步适应作为基于 RNA 的治疗剂在体内靶向转录亚型。