Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens.
Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens.
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DOI：
10.1186/s13059-020-02173-2
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发表时间：
2020-10-15
期刊：
影响因子：
12.3
通讯作者：
Hart T
中科院分区：
文献类型：
--
作者：
Dede M;McLaughlin M;Kim E;Hart T
在数百个细胞系中进行的合并文库CRISPR/Cas9敲除筛选已经确定了那些破坏导致适应性缺陷的基因，这是确定候选癌症靶点的关键步骤。然而，从这些单基因敲除筛选中检测到的必需基因的数量与细胞中组成型表达的基因的数量相比是低的。通过对癌症相关图谱生成的癌细胞系中筛选数据的系统分析，我们观察到所有组成型表达基因中有一半从未在任何CRISPR筛选中检测到，并且这些从未必需过的基因高度富集了旁系同源物。我们使用CRISPR/enCas 12 a双基因敲除筛选在三个细胞系中研究了大约400个候选parabolic对之间的功能缓冲。我们观察到24个合成的致命parabetes对，已经逃脱了检测单基因敲除筛选在严格的阈值。24种合成致死相互作用中的19种（79%）存在于三种细胞系中的至少两种中，24种中的14种（58%）存在于所有三种测试的细胞系中，包括稳定蛋白复合物的交替亚基以及功能冗余酶。总之，这些观察结果强烈表明，功能冗余旁系同源物代表了一组可靶向的遗传依赖性，这些遗传依赖性在基于单基因CRISPR的功能丧失筛选中在细胞必需基因中系统性地代表不足。
Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell. Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.