Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75–99%) and silencing (76–92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrP^C, screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and ‘post-pooling’ individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.

阵列 CRISPR 文库将基因扰动筛选的范围扩展到非选择性细胞表型。然而，文库生成需要组装数千个表达单向导 RNA （sgRNA） 的载体。在这里，通过利用大规模平行质粒克隆方法，我们表明可以构建阵列文库用于人类蛋白质编码基因的全基因组消融（19,936 个质粒）以及它们的激活和表观遗传沉默（22,442 个质粒），每个质粒编码一个由四个不重叠的 sgRNA 组成的阵列，旨在耐受大多数人类 DNA 多态性。四元 sgRNA 文库在缺失 （75-99%） 和沉默 （76-92%） 实验中产生了很高的扰动效率，并且在激活实验中产生了显著的倍数变化。此外，对 1,634 个人类转录因子的阵列激活筛选发现了细胞朊病毒蛋白 PrP^C 的 11 种新型调节因子，使用消融文库的混合版本进行筛选导致鉴定出 5 种新的自噬修饰物，否则这些修饰物不会被检测到，并且“混合后”单独产生的慢病毒消除了模板转换伪影并增强了用于表观遗传沉默的混合筛选的性能。Quadruple-sgRNA 阵列文库是一种强大的多功能资源，可用于靶向全基因组扰动。