Efficient gene knockout and genetic interactions: the IN4MER CRISPR/Cas12a multiplex knockout platform
Year: 2023
Type: preprint
Abstract: Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for combinatorial 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 approaches 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 Cas12a platform provides superior sensitivity and assay replicability. We demonstrate that Cas12a can independently target up to four genes from a single guide array, and we build on this knowledge by constructing a genome-scale library that expresses arrays of four guides per clone, a platform we call 'in4mer'. Our genome-scale human library, with only 49k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than four thousand paralog pairs, triples, and quads. Proof of concept screens in four 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/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.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: Bristol-Myers Squibb, Pfizer, GlaxoSmithKline, Cancer Prevention and Research Institute of Texas, Maze Therapeutics, Servier, National Institutes of Health, University of Texas MD Anderson Cancer Center, National Cancer Institute, National Institute of General Medical Sciences, Moonshot Research and Development Program, Cancer Moonshot
Awards: G10598100, Multiplex CRISPR genetic modeling of polypharmacology for precision medicine, Advanced tools for HCMI model genetic perturbation and metastasis characterization, G640481018, G7534854087, Deciphering the hierarchical modularity of the mammalian cell through network integration and complex genetic perturbation strategies, Identifying and targeting collateral lethal vulnerabilities in cancers, Cancer Center Support Grant, Expanding the known coding genome: identifying biological function for novel tORFs
OpenAlex ID: W4313535506