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

• 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
• 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

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 表征转录本同工型细胞功能在健康和疾病中的主要挑战之一是缺乏 专门有效地下调其表达的方法。靶向RNA的RNA引导VI CRISPR/CAS13系统构成了一种最近开发的用于敲低转录本的工具。他们的 通过与单链RNA互补的CRISPR RNA指南（GRNA）的结合来激活RNase活性 目标。迄今已测试的CRISPR/CAS13系统已被证明可以实现高度具体的敲低 内源性转录本，具有最小的脱靶效应，优于RNAi等电流方法。 虽然已显示CRISPR/CAS13在针对前MRNA的目标时有效地工作 通过靶向成熟mRNA中的独特连接的策略，可以允许敲低成绩单同工型 分子尚未探索。选择最佳的GRNA序列对于成功和特定的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 用CRISPR/CAS13D（最小的CAS13效应器）在成熟的mRNA分子中跨越外显子 - 外观连接 有效地将转录本表达降低到80％。此外，针对同工型特异性连接允许其 个人敲低而不会影响非靶向同工型。这些结果表明没有空间 靶向成熟mRNA分子中的连接的约束。肿瘤和癌细胞的转录组分析 线已经全面描述了替代转录本同工型的表达水平和身份。在 可行的许多研究表明，剪接失调是癌症的标志。因果关系 在转录本同工型表达和与癌症相关的表型之间尚未进行彻底研究。这里， 我们将扩展CRISPR/CAS13系统的适用性 癌症1）将大规模实验数据与机器学习方法相结合，以定义规则 针对特定的成绩单连接时GRNA设计2）优化管道和计算分析 在正向转录组合的汇总屏幕中使用CRISPR/CAS13系统需要询问细胞 转录本同工型的功能。通过扩大CRISPR/CAS13系统的应用，我们的管道将 提供在A中询问转录本同工型函数所需的分子工具和计算分析 强大的，公正的和高度可扩展的方式。我们希望我们的CRISPR/CAS13方法能够 克服当前方法在识别细胞特异性同工型表达和/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 在体内执行转录本同工型作为基于RNA的治疗。