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.

中文翻译：

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通过实验设计的sgRNA文库以高灵敏度进行基因组规模的CRISPR筛选

近年来，使用CRISPR / Cas9系统进行大规模遗传筛选已成为可扩展的方法，能够在各种生物学背景下以前所未有的效率和特异性来询问基因功能。通过这种方式，已经研究了对不同生物中多种细胞类型的蛋白质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，并且最佳实验实施和文库组成的标准仍在不断涌现。鉴于其在功能注释基因组中的广泛用途，基因组规模的CRISPR筛选的应用和解释将受益于一致且最佳的设计标准。我们报告了在进行Cas9单细胞克隆筛选中的生存力筛选方面的优势，而与Cas9大量人群相反。我们进一步系统地分析了人类细胞中已发表的CRISPR筛选，以鉴定具有一致的高靶向活性和低脱靶活性的单向导（sg）RNA。选定的指南收集在一个新颖的基因组规模的sgRNA库中，该库可有效识别核心和背景相关的必需基因。我们展示了凭经验设计的文库与优化的实验设计相结合如何在减少文库覆盖率的情况下增加基因必需性筛选的动态范围。