Minimized double guide RNA libraries enable scale-limited CRISPR/Cas9 screens

Genetic screens based on CRISPR/Cas technology are a powerful tool for understanding cellular phenotypes. However, the sequencing coverage and replicate requirements result in large experiment sizes, which are limiting when samples are scarce, or the protocols are expensive and laborious. Here, we present an approach to reduce the scale of genome-wide perturbation screens up to fivefold compared to conventional methods, without sacrificing performance. To do so, we deliver two randomly paired gRNAs into each cell, and rely on recent advances in gRNA design, and availability of gRNA effect measurements to reduce the number of gRNAs per gene. The paired gRNAs are expressed from separate cassettes that lead to gene perturbations both in isolation and in combination. We designed a genome-wide library that has effective size of 30,000 constructs, yet targets each gene with three gRNAs. Our double guide library gives similar results to a conventional single gRNA one, but with a substantially smaller cell culture volume. We demonstrate that genome-wide screens can be optimized in a demanding model such as induced pluripotent stem cells, reducing reagent cost 70% per replicate compared to standard approach, while retaining high performance. The screen design and the reduction in scale it provides will enable functional genomics experiments across many possible combinations of environments and genetic backgrounds, as well as in hard to culture primary cells.