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.

抽象的 在癌症中已经发现了数十万个突变。但是，绝大多数癌症 突变缺乏功能性生物学特征。因此，对它们对基因的影响知之甚少 在计算机预测中的功能超出了功能。开发实验模型来研究 这些突变是一个艰巨的挑战。我们开发了一种称为转录录的单个单元的技术 CRISPR测序（TISCC-SEQ），可在单细胞分辨率下对癌症突变进行建模。 CRISPR 工程将癌症基因突变引入细胞。单细胞RNA测序（SCRNA-SEQ）是功率 评估癌症基因组特征的工具。通过结合这两种方法，tiscc-seq具有 实验性癌症模型的层压化和可伸缩性的急剧增加的潜力。我们使用DNA 基础编辑者将特定的癌症突变引入单个细胞之间的靶基因。单分子 cDNA靶标的纳米孔测序直接识别每个细胞中的突变。通过整合单细胞 长和短读序列数据，每个细胞新引入的突变与同一细胞的基因匹配 表达数据。我们将开发TISCC-Seq作为一种新的用于工程癌的单细胞基因组平台 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遗传改变类型。对于目标1，我们将 开发用于在单细胞分辨率下对取代突变的高度多重功能筛选的TISCC-SEQ 在将突变基因型与同一单细胞的转录组相匹配的同时。我们将确定报道的癌症 具有已知生物学效应的突变以及其他未在结直肠或胃中鉴定的突变 癌症。接下来，我们将使用基本编辑方法来确定这些突变中的哪些可以设计。然后， 我们将提供基本编辑器和引导RNA，以设计不同单元的最多500个替代突变 线条和类器官。编辑后，这些单元将使用短和长阅读平台进行SCRNA-SEQ。 这些数据集将集​​成以提供单个单元突变与匹配的单个读数 相应的细胞的基因表达。 替代剪接越来越被认为是癌症的重要特征。一些基于顺式的癌症 突变发生在导致mRNA替代剪接的外显子内交界处。对于目标2，我们将发展 tiscc-seq是评估这类突变的方法。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道的，在外显子 - 内交界处具有基于CIS的突变。我们将 提高此过程的可伸缩性，以便可以并行研究此类突变的至少500个 使用集成的长读取测序。这些突变将在不同的细胞中引入 线条和类器官。总体而言，我们将开发一种新的CRISPR基因组技术，用于高度多重建模 单细胞分辨率和研究其生物学作用时的癌症突变。