Journal Article

Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform

Lin, Chenchu, Steger, Ryan, Ma, Xingdi, Doench, John G., Hart, Traver, Sangree, Annabel K., Esmaeili Anvar, Nazanin, Wilson, Lori L., Colic, Medina, Wang, Sidney H.

2024

Overview

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. Paralog synthetic lethals have been assessed with multiple CRISPR-based methods, but systematic comparison among these platforms is unavailable. Here, the authors systematically compare combinatorial perturbation platforms and establish the in4mer CRISPR/Cas12a multiplex knockout platform.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 13/106 / 38/43 / 45/41 / 45/77 / 49/109 / 49/22 / 49/23 / 49/47 / 631/114/2163 / 631/337/4041/3196 / 631/553/2690 / 631/61/191 / Bacterial Proteins / Cell Line, Tumor / Combinatorial analysis / CRISPR / CRISPR-Associated Proteins - genetics / CRISPR-Associated Proteins - metabolism / CRISPR-Cas Systems / Endodeoxyribonucleases / Epistasis, Genetic / Gene Knockout Techniques - methods / Gene Library / Genes / Genes, Essential / Genetic screening / Genomes / Genotypes / HEK293 Cells / Humanities and Social Sciences / Humans / Libraries / Mammalian cells / multidisciplinary / Multiplexing / Perturbation / Phenotypes / Platforms / RNA, Guide, CRISPR-Cas Systems - genetics / Science / Science (multidisciplinary)