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

项目摘要 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，这种异质性是一种主要的疾病。 对理解致癌机制和治疗的障碍。癌症在表观遗传学上各不相同， 在多个长度和时间尺度上以及在患者之间遗传。可能有许多不同的机制 甚至在单个癌细胞内也能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多个并发症的角度来理解癌症 基因和蛋白质。通过全基因组关联研究和epiGWAS识别的风险变体可以 在由许多384孔板组成的大型实验中通过RNAi和CRISPR单独扰动 文库，甚至可以使用条形码化策略在“一锅法”中组合扰动和筛选。 然而，即使是最先进的CRISPR筛选方法也限于2或3个功能扰动。 每一个细胞都有变异。这些限制来自gRNA阵列中重复序列的困难 存在于表达构建体合成和稳定性两者中。在这里，我们提出了一种新的方法， 能够表达数千种不同gRNA的随机化组合文库，每个细胞都有一个 表达超过30种gRNA的阵列的群体。

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.