Department of Cell Biology, Duke University, Durham, NC, USA.
Department of Biomedical Engineering, Duke University, Durham, NC, USA.
Methods Mol Biol. 2022;2485:1-13. doi: 10.1007/978-1-0716-2261-2_1.
CRISPR-Cas9-based screening technologies enable precise, high-throughput genetic and epigenetic manipulation to study mechanisms of development and disease and identify new therapeutic targets. Here, we describe a general protocol for the generation of custom, pooled CRISPR sgRNA libraries for screening in cardiomyocyte cultures. This methodology can address a variety of lab-specific research questions in cardiomyocytes and other cell types, as the genes to be modified can be curated or whole genomes can be investigated. The use of lentiviral sgRNA delivery followed by high-throughput sequencing allows for rapid comparison and identification of candidate genes and epigenetic modifiers, which can be further validated individually or in sub-pooled libraries following screening.
基于 CRISPR-Cas9 的筛选技术可实现精确、高通量的遗传和表观遗传操作，用于研究发育和疾病的机制，并确定新的治疗靶点。在这里，我们描述了一种用于在心肌细胞培养物中进行筛选的定制、 pooled CRISPR sgRNA 文库的生成的通用方案。该方法可解决心肌细胞和其他细胞类型中的各种特定于实验室的研究问题，因为可以对要修饰的基因进行策展或研究整个基因组。慢病毒 sgRNA 递送至高通量测序允许快速比较和鉴定候选基因和表观遗传修饰物，随后可以在筛选后单独或在亚池化文库中进一步验证这些基因和修饰物。
