Single cell modeling of cancer mutations

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 突变缺乏功能性生物学特征。因此，关于它们对基因的影响知之甚少。 功能超出计算机预测。开发实验模型以研究 这些突变是一项艰巨的挑战。我们开发了一种叫做转录单细胞的技术 CRISPR测序（TISCC-seq），以单细胞分辨率提供癌症突变的建模。CRISPR 基因工程将癌症基因突变引入细胞。单细胞RNA测序（scRNA-seq）是一种强大的 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 潜在的并行化和实验癌症模型的可扩展性显着增加。我们使用DNA 碱基编辑器将特定的癌症突变引入单个细胞中的靶基因。单分子 cDNA靶的纳米孔测序直接鉴定每个细胞中的突变。通过整合单细胞 长读和短读序列数据，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作为工程癌症的新单细胞基因组平台 突变到细胞系和原代组织培养物中。 单碱基突变是最常见的癌症遗传改变类型。目标1： 开发TISCC-seq，用于以单细胞分辨率进行置换突变的高度多重功能筛选 同时将突变基因型与同一个单细胞的转录组相匹配。我们将确定报告的癌症 具有已知生物学效应的突变和在结肠直肠或胃肠道中未鉴定的其他突变 癌接下来，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改造。然后， 我们将提供碱基编辑器和指导RNA，在不同的细胞中设计多达500个取代突变， 线和类器官。编辑后，细胞将经历具有短读段和长读段平台的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细胞突变相匹配。 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。一些顺式癌症 突变发生在外显子-内含子连接处，导致mRNA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外显子-内含子连接处有顺式突变。我们将 增加该过程的可扩展性，以便可以并行研究至少500个此类突变 使用整合的长和短读取测序。这些突变将被引入不同的细胞 线和类器官。总的来说，我们将开发一种新的CRISPR基因组技术，用于高度多重建模 癌症突变的单细胞分辨率和研究其生物学效应。

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.