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

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的方法，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。

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.