Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform.
Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform.
复制标题
使用 in4mer CRISPR/Cas12a 多重敲除平台进行高效基因敲除和遗传相互作用筛选。
DOI：
10.1038/s41467-024-47795-3
复制
发表时间：
2024
影响因子：
16.6
通讯作者：
Hart,Traver
中科院分区：
文献类型：
--
作者：
EsmaeiliAnvar,Nazanin;Lin,Chenchu;Ma,Xingdi;Wilson,LoriL;Steger,Ryan;Sangree,AnnabelK;Colic,Medina;Wang,SidneyH;Doench,JohnG;Hart,Traver
遗传相互作用介导表型从基因型的出现，但在哺乳动物细胞中的组合遗传扰动技术具有挑战性的规模。在这里，我们从先前的CRISPR遗传相互作用筛选中鉴定出背景独立的副作用合成致死，并发现Cas 12 a平台提供了上级灵敏度和测定可重复性。我们开发了in 4 mer Cas 12 a平台，该平台使用靶向相同或不同基因的四个独立指导RNA的阵列。我们构建了一个基因组规模的文库，Inzolia，比典型的CRISPR/Cas9文库小约30%，同时也靶向约4000个碱基对。在癌细胞中的筛选证明了与CRISPR/Cas9文库相似的核心和背景依赖性必需基因的区分，以及各种家族大小的旁系同源物之间的合成致死和掩蔽/缓冲遗传相互作用的检测。重要的是，与其他遗传相互作用方法相比，in 4 mer平台提供了五倍的文库大小减少，大大降低了这些测定所需的成本和工作量。
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.