PDF(Pubmed)
Abstract：
Reverse genetics offers precise functional insights into genes through the targeted manipulation of gene expression followed by phenotypic assessment. While these approaches have proven effective in model organisms such as Saccharomyces cerevisiae, large-scale genetic manipulations in human cells were historically unfeasible due to methodological limitations. However, recent advancements in functional genomics, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-based screening technologies and next-generation sequencing platforms, have enabled pooled screening technologies that allow massively parallel, unbiased assessments of biological phenomena in human cells. This review provides a comprehensive overview of cutting-edge functional genomic screening technologies applicable to human cells, ranging from short hairpin RNA screens to modern CRISPR screens. Additionally, we explore the integration of CRISPR platforms with single-cell approaches to monitor gene expression, chromatin accessibility, epigenetic regulation, and chromatin architecture following genetic perturbations at the omics level. By offering an in-depth understanding of these genomic screening methods, this review aims to provide insights into more targeted and effective strategies for genomic research and personalized medicine.
摘要：
反向遗传学通过有针对性地操纵基因表达，然后进行表型评估，为基因提供了精确的功能见解。虽然这些方法已被证明在酿酒酵母等模型生物中有效，由于方法学上的限制，人类细胞中的大规模遗传操作在历史上是不可行的。然而,功能基因组学的最新进展，特别是基于CRISPR的筛选技术和下一代测序平台，已经实现了允许大规模并行的汇集筛查技术，人类细胞中生物学现象的无偏见评估。这篇综述全面概述了适用于人类细胞的尖端功能基因组筛选技术，从shRNA屏幕到现代CRISPR屏幕。此外,我们探索了CRISPR平台与单细胞方法的整合来监测基因表达，染色质可及性，表观遗传调控，以及组学水平遗传扰动后的染色质结构。通过提供对这些基因组筛选方法的深入了解，这篇综述旨在为基因组研究和个性化医疗提供更有针对性和有效的策略。
