Klann Tyler S, Crawford Gregory E, Reddy Timothy E, Gersbach Charles A
Department of Biomedical Engineering, Duke University, Durham, NC, USA.
Center for Genomic and Computational Biology, Duke University, Durham, NC, USA.
Methods Mol Biol. 2018;1767:447-480. doi: 10.1007/978-1-4939-7774-1_25.
Genomic regulatory elements that control gene expression play an important role in many traits and diseases. Identifying the regulatory elements associated with each gene or phenotype and understanding the function of that element remain a significant challenge. To address this technological need, we developed CRISPR/Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. This protocol includes detailed instructions for design and cloning of gRNA libraries, construction of endogenous reporter cell lines via CRISPR/Cas9-mediated knock-in of fluorescent proteins, overall screen design, and recovery of the gRNA library for enrichment analysis. This protocol will be generally useful for implementing genome engineering technologies for high-throughput functional annotation of putative regulatory elements in their native chromosomal context.
控制基因表达的基因组调控元件在许多性状和疾病中发挥着重要作用。识别与每个基因或表型相关的调控元件并了解该元件的功能仍然是一项重大挑战。为满足这一技术需求，我们开发了基于CRISPR/Cas9的表观基因组调控元件筛选（CERES）方法，以改进在天然基因组环境中对调控元件活性的高通量筛选。本方案包括gRNA文库设计和克隆、通过CRISPR/Cas9介导的荧光蛋白敲入构建内源性报告细胞系、整体筛选设计以及回收gRNA文库进行富集分析的详细说明。本方案对于在其天然染色体环境中对推定调控元件进行高通量功能注释的基因组工程技术的实施将具有普遍用途。
