Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform
Year: 2024
Type: article
Abstract: Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.
Source: Nature Communications
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,790
Funders: U.S. Department of Health and Human Services, Cancer Prevention and Research Institute of Texas, 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, Identification of collateral lethal and synthetic lethal targets in pancreatic and colorectal cancers, Multiplex CRISPR genetic modeling of polypharmacology for precision medicine, G4365555424, G640481018, G686819199, 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: W4395695320