Major efforts on pooled library CRISPR knockout screening across hundreds of cell lines have 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 are very low compared to the number of constitutively expressed genes in a cell, raising the question of why there are so few essential genes. Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observed that half of all constitutively-expressed genes are never hits in any CRISPR screen, and that these never-essentials are highly enriched for paralogs. We investigated paralog buffering through systematic dual-gene CRISPR knockout screening by testing algorithmically defined ~400 candidate paralog pairs with the enCas12a multiplex knockout system in three cell lines. We observed 24 synthetic lethal paralog pairs which have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions were present in at least two out of three cell lines and 14 of 24 (58%) were 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 paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes due to genetic buffering in monogenic CRISPR-based mammalian functional genomics approaches.

中文翻译：

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多重enCas12a筛选显示全基因组CRISPR / Cas9敲除筛选系统性地缺乏通过旁系同源物进行功能缓冲

在数百种细胞系的库文库CRISPR基因敲除筛选中的主要努力已鉴定出其基因断裂导致适应性缺陷的基因，这是鉴定候选癌症靶标的关键步骤。但是，与细胞中组成型表达的基因数量相比，从这些单基因敲除筛选中检测到的必需基因数量非常低，这引发了一个问题，即为什么必需基因如此之少。通过对癌症依赖性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性表达基因中有一半从未在任何CRISPR筛选中被点击，并且这些从不必需的基因对于旁系同源物高度丰富。我们通过在三个细胞系中使用enCas12a多重敲除系统测试算法定义的〜400个候选旁系对，通过系统化的双基因CRISPR敲除筛选研究了旁系同源缓冲。我们观察到了24个合成致死旁系同源物对，它们已在严格的阈值下逃脱了单基因敲除筛选的检测。在所有三个测试的细胞系中，至少有两个中有19个（24％）（24％）合成致死性相互作用，在所有三个测试细胞系中都存在24个中的14个（58％），包括稳定蛋白复合物的替代亚基以及功能上多余的酶。