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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：
9795162
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/9795162
关键词：
Address Automobile Driving 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.

项目总结