German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and Heidelberg University, BioQuant and Medical Faculty Mannheim, D-69120, Heidelberg, Germany.
BMC Biol. 2020 Nov 23;18(1):174. doi: 10.1186/s12915-020-00905-1.
In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding and noncoding genome of numerous cell types in different organisms have been interrogated. However, screening designs vary greatly and criteria for optimal experimental implementation and library composition are still emerging. Given their broad utility in functionally annotating genomes, the application and interpretation of genome-scale CRISPR screens would greatly benefit from consistent and optimal design criteria.
We report advantages of conducting viability screens in selected Cas9 single-cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg) RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a novel genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes.
We show how empirically designed libraries in combination with an optimized experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.
近年来，基于 CRISPR/Cas9 系统的大规模基因筛选技术已经成为一种可扩展的方法，能够以空前的效率和特异性在各种生物环境中研究基因功能。通过这种方法，已经研究了许多不同生物体中各种细胞类型的蛋白编码和非编码基因组的功能依赖性。然而，筛选设计差异很大，最佳实验实施和文库组成的标准仍在不断出现。鉴于它们在功能注释基因组方面的广泛应用，基于 CRISPR 的全基因组筛选的应用和解释将极大地受益于一致和最佳的设计标准。
我们报告了在选定的 Cas9 单细胞克隆中进行生存能力筛选相对于 Cas9 批量群体的优势。我们进一步系统地分析了已发表的人类细胞中的 CRISPR 筛选，以确定具有一致的高靶标和低脱靶活性的单引导（sg）RNA。选定的指南被收集在一个新的全基因组 sgRNA 文库中，该文库可有效地鉴定核心和上下文相关的必需基因。
我们展示了如何通过经验设计的文库与优化的实验设计相结合，在降低文库覆盖率的情况下，提高基因必需性筛选的动态范围。
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