Combined genome-scale fitness and paralog synthetic lethality screens with just 44k clones: the IN4MER CRISPR/Cas12a multiplex knockout platform.

Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for multiplex genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different CRISPR/Cas multiplexing technologies and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the CRISPR/enCas12a platform provides superior sensitivity and assay replicability. We demonstrate that enCas12a can independently target up to four genes from a single guide array, and build on this knowledge by constructing a one-component library that expresses arrays of four guides per clone, a platform we call 'in4mer'. Our genome-scale human library, with only 44k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than two thousand paralog pairs, triples, and quads. Proof of concept screens in two cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state of the art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking (also known as buffering) genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.