CRISPR-Cas9 筛选揭示了人类癌细胞系中常见的重要 miRNA。

IF 10.4 1区 生物学 Q1 GENETICS & HEREDITY
Daniel J Merk, Linda Paul, Foteini Tsiami, Helen Hohenthanner, Ghazal Mohseni Kouchesfahani, Lara A Haeusser, Bianca Walter, Adam Brown, Nicole S Persky, David E Root, Ghazaleh Tabatabai
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引用次数: 0

摘要

背景:利用CRISPR-Cas9系统进行全基因组功能筛选是发现癌症细胞系中肿瘤特异性和共同遗传依赖性的有力工具。然而,目前的 CRISPR-Cas9 基因敲除文库主要针对蛋白编码基因,这限制了基于功能基因组学的 miRNA 功能研究。这限制了基于功能基因组学的 miRNA 功能研究:方法:我们设计了一种新型 CRISPR-Cas9 基因敲除文库(lentiG-miR),其中包含 8107 个不同的 sgRNA,靶向总共 1769 个人类 miRNA。我们利用代表 16 种不同肿瘤实体的 45 种人类癌症细胞系进行了负选择筛选,以确定 miRNA 适合基因。采用监督和非监督本质分类器相结合的方法对每个细胞系中的适合度 miRNA 进行评分。使用第 90 百分位数法确定了不同癌症细胞系中共同的基本 miRNA。为了进行后续验证,我们在不同的癌细胞系中对选定的常见基本 miRNA 进行了基因敲除实验,并进行了基因表达谱分析:结果:我们发现,与之前描述的 miRNA 靶向文库相比,lentiG-miR 的蛋白编码基因脱靶活性明显降低,miRNA 基因覆盖率更高,同时保留了较高的靶向活性。一小部分 miRNA 显示出了靶向 sgRNA 的稳健耗竭,而且我们观察到靶向同一 miRNA 基因的冗余 sgRNA 具有高度的一致性。在 45 个人类癌细胞系中,所有靶向的人类 miRNA 中只有 217 个(12%)在至少一个模型中作为适配基因得分,而且大多数 miRNA 的适配效应仅限于细胞系的一小部分。与此相反,我们发现了 49 种常见的基本 miRNA,这些 miRNA 在绝大多数细胞系中都具有相同的适应性特征。转录谱分析证实,在不同的癌症细胞系中,基因表达的变化与基因敲除单个常见基本 miRNA 的反应高度一致:我们的研究提出了一种 miRNA 靶向 CRISPR-Cas9 基因敲除文库,它具有高基因覆盖率和优化的靶上和靶下活性。利用 lentiG-miR 文库的优势,我们确定了人类癌细胞系中的 miRNA 适应基因目录,为进一步研究人类癌症中的 miRNA 奠定了基础。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
CRISPR-Cas9 screens reveal common essential miRNAs in human cancer cell lines.

Background: Genome-wide functional screening using the CRISPR-Cas9 system is a powerful tool to uncover tumor-specific and common genetic dependencies across cancer cell lines. Current CRISPR-Cas9 knockout libraries, however, primarily target protein-coding genes. This limits functional genomics-based investigations of miRNA function.

Methods: We designed a novel CRISPR-Cas9 knockout library (lentiG-miR) of 8107 distinct sgRNAs targeting a total of 1769 human miRNAs and benchmarked its single guide RNA (sgRNA) composition, predicted on- and off-target activity, and screening performance against previous libraries. Using a total of 45 human cancer cell lines, representing 16 different tumor entities, we performed negative selection screens to identify miRNA fitness genes. Fitness miRNAs in each cell line were scored using a combination of supervised and unsupervised essentiality classifiers. Common essential miRNAs across distinct cancer cell lines were determined using the 90th percentile method. For subsequent validation, we performed knockout experiments for selected common essential miRNAs in distinct cancer cell lines and gene expression profiling.

Results: We found significantly lower off-target activity for protein-coding genes and a higher miRNA gene coverage for lentiG-miR as compared to previously described miRNA-targeting libraries, while preserving high on-target activity. A minor fraction of miRNAs displayed robust depletion of targeting sgRNAs, and we observed a high level of consistency between redundant sgRNAs targeting the same miRNA gene. Across 45 human cancer cell lines, only 217 (12%) of all targeted human miRNAs scored as a fitness gene in at least one model, and fitness effects for most miRNAs were confined to small subsets of cell lines. In contrast, we identified 49 common essential miRNAs with a homogenous fitness profile across the vast majority of all cell lines. Transcriptional profiling verified highly consistent gene expression changes in response to knockout of individual common essential miRNAs across a diverse set of cancer cell lines.

Conclusions: Our study presents a miRNA-targeting CRISPR-Cas9 knockout library with high gene coverage and optimized on- and off-target activities. Taking advantage of the lentiG-miR library, we define a catalogue of miRNA fitness genes in human cancer cell lines, providing the foundation for further investigation of miRNAs in human cancer.

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来源期刊
Genome Medicine
Genome Medicine GENETICS & HEREDITY-
CiteScore
20.80
自引率
0.80%
发文量
128
审稿时长
6-12 weeks
期刊介绍: Genome Medicine is an open access journal that publishes outstanding research applying genetics, genomics, and multi-omics to understand, diagnose, and treat disease. Bridging basic science and clinical research, it covers areas such as cancer genomics, immuno-oncology, immunogenomics, infectious disease, microbiome, neurogenomics, systems medicine, clinical genomics, gene therapies, precision medicine, and clinical trials. The journal publishes original research, methods, software, and reviews to serve authors and promote broad interest and importance in the field.
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