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使用深度突变扫描和基础编辑进行变异函数测量的并排比较

Ivan Sokirniy¹, Haider Inam^1,2, Marta Tomaszkiewicz^1,2

¹Huck Institute for the Life Sciences, University Park, PA 16802.

bioRxiv : the preprint server for biology

|July 15, 2024

概括

深度突变扫描 (DMS) 和CRISPR基编辑 (BE) 显示了变异注释的强烈相关性. 优化BE方法,比如过sgRNAs,可以提高功能基因组学研究的准确性.

科学领域:

哺乳动物功能基因组学
基于CRISPR编辑技术的基础编辑技术.
高通量选的高通量选

背景情况:

变异注释对于理解基因功能至关重要.
深度突变扫描 (DMS) 是一种标准方法,但CRISPR基编辑 (BE) 提供了一个有前途的替代方案.
在变体注释中对BE的直接比较和验证需求尚不清楚.

研究的目的:

为了直接比较深度突变扫描 (DMS) 和CRISPR基编辑 (BE) 的变异注释.
评估基于BE的变异函数注释所需的实验验证.
优化BE查策略,以提高数据与DMS的一致性.

主要方法:

在同一个细胞系中执行并行DMS和BE屏幕.
对BE数据应用过策略,重点关注高效的sgRNA和单次编辑结果.
通过在聚合屏幕中直接测量编辑的变体来量化变体效应.

主要成果:

通过选择高效的sgRNA和专注于可能的编辑来优化BE屏幕,显著改善了与DMS数据的一致性.
诱导单次编辑的sgRNAs的一个简单的过器使得从聚合的sgRNA测序中实现直接变异注释.
在聚合的BE屏幕中测量实际变异,而不是sgRNA丰度,恢复了高质量的注释数据,即使使用多次编辑指南.

结论:

克里斯普尔基编辑 (BE) 显示出高程度的相关性与金级标准深度突变扫描 (DMS) 变体注释.
战略过和直接变异测量提高了BE在功能基因组学中的可靠性和效率.
BE是大规模人类基因变异注释的可行和有效替代方案.

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