Intracellular CRISPR gRNA assembly for massively multiplexed, one pot, (epi)genetic screening

用于大规模多重、一锅、（表观）遗传筛选的细胞内 CRISPR gRNA 组装

• 批准号: 10242748
• 负责人: Albert Keung
• 金额: $ 16.53万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242748
• 关键词: Address; Automobile Driving; Bar Codes; CRISPR library; CRISPR screen; Cancer Biology; Cancer Etiology; Cancer cell line; Cancerous; Cells; Cellular immunotherapy; ChIP-seq; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Developmental Biology; Dideoxy Chain Termination DNA Sequencing; Dimensions; Disease; Engineering; Epigenetic Process; Etiology; Face; Gene Proteins; Genes; Genetic; Genetic Screening; Genetic Variation; Genome; Guide RNA; Heterogeneity; Human; In Situ; Individual; Length; Libraries; Ligation; Malignant Neoplasms; Measurement; Measures; Methods; Mutation; Oncogenic; Patients; Periodicity; Population; Process; Property; RNA; RNA Interference; Randomized; Repetitive Sequence; Research Personnel; Resistance; Sampling; System; Testing; Time; Tissues; Variant; Work; anticancer research; bisulfite sequencing; cancer risk; combinatorial; disease heterogeneity; epigenetic variation; epigenome-wide association studies; experience; experimental study; genome wide association study; high dimensionality; homologous recombination; knock-down; metastatic process; new technology; next generation sequencing; risk variant; scaffold; screening; stem cell biology; synthetic biology; tumor

Project Summary It has been clear for over a decade that cancer is not a single disease and that this heterogeneity is a primary barrier to the understanding of oncogenic mechanisms and treatments. Cancers vary epigenetically and genetically at multiple length and time scales and between patients. There can be many distinct mechanisms driving oncogenic and metastatic processes, even within a single cancerous cell. The challenge researchers and clinicians face is how to understand cancer from the perspective of simultaneous perturbations to multiple genes and proteins. Risk variants identified through genome wide association studies and epiGWAS can be individually perturbed in large experiments comprised of many 384-well plates through RNAi and CRISPR libraries, and even be combinatorially perturbed and screened in `one-pot' using barcoding strategies. However, even state-of-the-art CRISPR screening methods are restricted to functional perturbations of 2 or 3 variants at a time per cell. These restrictions arise from the difficulties repetitive sequences in gRNA arrays present in both expression construct synthesis and stability. Here we propose a new method that will be capable of expressing randomized combinatorial libraries of thousands of distinct gRNAs, with each cell of a population expressing an array of over 30 gRNAs.

项目概要 十多年来，人们已经清楚地认识到癌症不是一种单一的疾病，这种异质性是导致癌症的主要因素。 阻碍理解致癌机制和治疗。癌症在表观遗传上有所不同 在多个长度和时间尺度以及患者之间进行遗传分析。可以有许多不同的机制 即使在单个癌细胞内，也会驱动致癌和转移过程。挑战研究人员和 临床医生面临的问题是如何从同时扰动多个因素的角度来理解癌症 基因和蛋白质。通过全基因组关联研究和 epiGWAS 识别的风险变异可以 通过 RNAi 和 CRISPR 在由许多 384 孔板组成的大型实验中单独扰动 文库，甚至可以使用条形码策略进行“一锅法”组合干扰和筛选。 然而，即使是最先进的 CRISPR 筛选方法也仅限于 2 或 3 的功能扰动。 每个细胞一次的变体。这些限制源于 gRNA 阵列中重复序列的困难 存在于表达构建体的合成和稳定性中。在这里，我们提出了一种新方法，即 能够表达数千个不同 gRNA 的随机组合文库，每个细胞具有 表达超过 30 个 gRNA 的群体。