Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.

中文翻译：

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使用混合 Cas9-Cas12a 平台的遗传相互作用作图和外显子分辨率功能基因组学。

遗传相互作用 (GI) 的系统映射和哺乳动物细胞中相当大的基因组片段的功能的询问代表了生物医学研究的重要目标。为了推进这些目标，我们提出了一种基于 CRISPR（成簇规律间隔的短回文重复序列）的组合遗传操作筛选系统，该系统采用 CRISPR 相关核酸酶 9 和 12a（Cas9 和 Cas12a）的共表达和机器学习优化的混合文库Cas9-Cas12a 指导 RNA。这个名为 Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA) 的系统优于单独使用 Cas9 或 Cas12a 编辑的基因筛选。CHyMErA 在哺乳动物旁系同源基因对消融中的应用揭示了广泛的 GI 并揭示了通常被功能冗余掩盖的表型。CHyMErA 在化学遗传学相互作用筛选中的应用可识别影响细胞生长以响应 mTOR 通路抑制的基因。此外，通过系统地针对数千个可变剪接事件，CHyMErA 识别出人类细胞系适应性的外显子。因此，CHyMERA 代表了一种有效的 GI 作图筛选方法和相当大的基因组区域（例如替代外显子）的功能分析。