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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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要十多年来，人们清楚地看到，癌症不是一种单一的疾病，这种异质性是主要的阻碍了对致癌机制和治疗方法的理解。癌症在表观遗传学上有所不同在遗传上，在多种长度和时间尺度上，在患者之间。可以有许多不同的机制推动致癌和转移过程，即使在单个癌细胞内也是如此。挑战研究人员和临床医生面临的问题是如何从多个同时扰动的角度来认识癌症基因和蛋白质。通过全基因组关联研究和表观遗传多样性研究确定的风险变异可以是通过RNAi和CRISPR在由多个384孔板组成的大型实验中单独扰动库，甚至被组合扰动，并使用条形码策略在“一锅”中进行筛选。然而，即使是最先进的CRISPR筛选方法也仅限于2或3的函数扰动每个细胞一次变异。这些限制源于gRNA阵列中重复序列的困难同时存在于表达结构的合成性和稳定性中。在这里，我们提出了一种新方法，它将是能够表达数千个不同gRNA的随机组合文库，其中一个细胞表达超过30个gRNA阵列的种群。