Single cell modeling of cancer mutations

癌症突变的单细胞建模

• 批准号: 10612689
• 负责人: Hanlee P Ji
• 金额: $ 37.53万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612689
• 关键词: Address; Alternative Splicing; Amino Acid Substitution; Base Sequence; Biological; Biological Sciences; CRISPR/Cas technology; Cancer Gene Mutation; Cancer Model; Categories; Cell Line; Cell model; Cells; Cellular Assay; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Colorectal Cancer; Complementary DNA; DNA; Data; Data Set; Detection; Engineering; Exons; Experimental Models; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic; Genetic Code; Genetic Transcription; Genomics; Genotype; Guide RNA; Individual; Introns; Length; Link; Malignant Neoplasms; Messenger RNA; Methods; Modeling; Mutation; Normal Cell; Oncogenes; Organoids; Pattern; Point Mutation; Process; Protein Isoforms; RNA Sequences; RNA Splicing; Reporting; Resolution; Somatic Mutation; Technology; Testing; Transcript; base; base editing; base editor; cancer cell; cancer type; cellular engineering; data integration; design; experimental study; gene function; genome editing; genomic platform; in silico; mRNA sequencing; malignant stomach neoplasm; mutation screening; nanopore; novel; novel strategies; nuclease; parallelization; screening; single cell sequencing; single molecule; single-cell RNA sequencing; technological innovation; tissue culture; tool; transcriptome

ABSTRACT Hundreds of thousands of mutations have been identified in cancer. However, the vast majority of cancer mutations lack functional biological characterization. Therefore, very little is known about their impact on gene function beyond in silico predictions. Developing experimental models to study the biological consequences of these mutations is a daunting challenge. We developed a technology called transcript-informed single cell CRISPR sequencing (TISCC-seq) that provides modeling of cancer mutations at single cell resolution. CRISPR engineering introduces cancer gene mutations into cells. Single-cell RNA sequencing (scRNA-seq) is a power tool for evaluating the genomic features of cancer. By combining these two approaches TISCC-seq has the potential for dramatic increases in parallelization and scalability of experimental cancer models. We use DNA base editors to introduce specific cancer mutations into target genes among individual cells. Single molecule nanopore sequencing of the cDNA target directly identifies the mutation in each cell. By integrating single cell long and short read sequence data, each cell’s newly introduced mutation is matched to the same cell’s gene expression data. We will develop TISCC-seq as a new single cell genomic platform for engineering cancer mutations into cell lines and primary tissue cultures. Single base mutations are the most commonly reported type of cancer genetic alteration. For Aim 1, we will develop TISCC-seq for highly multiplexed functional screening of substitution mutations at single cell resolution while matching the mutation genotype to the same single cell’s transcriptome. We will identify reported cancer mutations with known biological effects and others which are not characterized identified in colorectal or gastric cancer. Next, we will determine which of these mutations can be engineered using base editing methods. Then, we will deliver base editors and guide RNAs to engineer up to 500 substitution mutations across different cell lines and organoids. Post-editing, the cells will undergo scRNA-seq with both short and long-read platform. These data sets will be integrated to provide a single readout where the single cell mutation is matched to the corresponding cell’s gene expression. Alternative splicing is increasingly recognized as an important feature of cancer. Some cis-based cancer mutations occur in exon-intron junctions that lead to alternative splicing of mRNAs. For Aim 2, we will develop TISCC-seq as a method to evaluate this category of mutations. First, we will identify a set of 100 cancer genes that have cis-based mutations at exon-intron junctions as reported in colorectal or gastric cancer. We will increase the scalability of this process such that at least 500 of this class of mutations can be studied in parallel using an integrated long and short read sequencing. These mutations will be introduced across different cell lines and organoids. Overall, we will develop a new CRISPR genomic technology for highly multiplexed modeling of cancer mutations at single cell resolution and studying their biological effects.

摘要 在癌症中已经发现了数十万种突变。然而，绝大多数癌症 突变缺乏功能生物学特征。因此，人们对它们对基因的影响知之甚少。 在硅胶预测中发挥更大的作用。开发实验模型来研究生物后果 这些突变是一个令人生畏的挑战。我们开发了一种名为转录信息单细胞的技术 CRISPR测序(TISCC-SEQ)，提供单细胞分辨率的癌症突变建模。CRISPR 工程学将癌症基因突变引入细胞。单细胞rna测序(scrna-seq)是一种力量 用于评估癌症基因组特征的工具。通过将这两种方法结合起来，TISCC-seq拥有 实验癌症模型的并行化和可伸缩性方面的潜力显著增加。我们使用DNA 碱基编辑将特定的癌症突变引入单个细胞中的靶基因。单分子 对cDNA靶的纳米孔测序直接确定每个细胞中的突变。通过集成单个电池 长短读取序列数据，每个细胞的新引入的突变与相同细胞的基因匹配 表达式数据。我们将开发TISCC-seq作为一种新的单细胞基因组工程癌症平台。 突变成细胞系和原代组织培养物。 单碱基突变是最常见的癌症基因改变类型。对于目标1，我们将 建立TISCC-SEQ用于在单细胞分辨率下对替代突变进行高度多元化的功能筛选 同时将突变基因与同一单细胞的转录组进行匹配。我们将确认已报告的癌症 具有已知生物学效应的突变和其他在结直肠或胃中未发现特征的突变 癌症。接下来，我们将确定这些突变中的哪些可以使用碱基编辑方法进行工程处理。然后, 我们将提供碱基编辑器并引导RNA在不同细胞中设计多达500个替换突变 线条和管风琴。编辑后，细胞将进行短读和长读平台的scRNA-seq。 这些数据集将被整合，以提供单个读数，其中单个细胞突变与 相应细胞的基因表达。 选择性剪接越来越被认为是癌症的一个重要特征。一些顺式细胞癌 突变发生在外显子-内含子连接中，导致mRNAs的选择性剪接。对于目标2，我们将开发 TISCC-seq作为评估这类突变的一种方法。首先，我们将确定一组100个癌症基因 在结直肠癌或胃癌中报告的在外显子-内含子连接具有顺式突变的基因。我们会 提高这一过程的可扩展性，以便至少可以并行研究这类突变中的500个 使用集成的长和短读取序列。这些突变将被引入不同的细胞 线条和管风琴。总体而言，我们将开发一种新的CRISPR基因组技术，用于高度多元化的建模 在单细胞分辨率下研究癌症突变，并研究它们的生物学效应。