Multiple-gene targeting and mismatch tolerance can confound analysis of genome-wide pooled CRISPR screens
Multiple-gene targeting and mismatch tolerance can confound analysis of genome-wide pooled CRISPR screens
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DOI：
10.1186/s13059-019-1621-7
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发表时间：
2019-01-25
期刊：
影响因子：
12.3
通讯作者：
Martin, Scott E.
中科院分区：
文献类型：
--
作者：
Fortin, Jean-Philippe;Tan, Jenille;Martin, Scott E.
背景使用 CRISPR/Cas9 系统进行全基因组功能丧失筛选可以有效发现癌细胞的脆弱性。虽然一些研究集中于纠正与拷贝数改变相关的 DNA 切割毒性偏差，但尚未讨论 CRISPR 筛选中共同靶向多个基因组位点的 sgRNA 的影响。结果在这项工作中，我们分析了来自 391 个癌细胞系的 CRISPR 必要性筛选数据，以表征多靶点 sgRNA 引起的偏差。我们研究了两种类型的多靶标：通过完美序列互补性预测的靶标和通过最多两个核苷酸错配的序列互补性预测的脱靶。我们发现，靶向和脱靶的数量都会以细胞系特异性的方式增加 sgRNA 活性，并且现有的基因敲除效应加性模型无法捕获共靶向基因之间可能发生的遗传相互作用。我们使用旁系同源基因之间的合成致死率来表明遗传相互作用可能会导致从多靶点 sgRNA 估计的重要性分数出现偏差。我们进一步表明，单错配耐受 sgRNA 可能会混淆基因必需性分析，并导致错误的共必需性功能网络。最后，我们还发现位于原型间隔区的单核苷酸多态性会因错配耐受性而损害靶向活性。结论我们展示了多靶点效应对估计癌细胞依赖性的影响以及sgRNA-DNA结合中错配耐受性引起的脱靶效应的影响。
BackgroundGenome-wide loss-of-function screens using the CRISPR/Cas9 system allow the efficient discovery of cancer cell vulnerabilities. While several studies have focused on correcting for DNA cleavage toxicity biases associated with copy number alterations, the effects of sgRNAs co-targeting multiple genomic loci in CRISPR screens have not been discussed.ResultsIn this work, we analyze CRISPR essentiality screen data from 391 cancer cell lines to characterize biases induced by multi-target sgRNAs. We investigate two types of multi-targets: on-targets predicted through perfect sequence complementarity and off-targets predicted through sequence complementarity with up to two nucleotide mismatches. We find that the number of on-targets and off-targets both increase sgRNA activity in a cell line-specific manner and that existing additive models of gene knockout effects fail at capturing genetic interactions that may occur between co-targeted genes. We use synthetic lethality between paralog genes to show that genetic interactions can introduce biases in essentiality scores estimated from multi-target sgRNAs. We further show that single-mismatch tolerant sgRNAs can confound the analysis of gene essentiality and lead to incorrect co-essentiality functional networks. Lastly, we also find that single nucleotide polymorphisms located in protospacer regions can impair on-target activity as a result of mismatch tolerance.ConclusionWe show the impact of multi-target effects on estimating cancer cell dependencies and the impact of off-target effects caused by mismatch tolerance in sgRNA-DNA binding.