Techniques for Validating CRISPR Changes Using RNA-Sequencing Data

Susan K. Rathe

doi:10.3390/genes16040369

2025-03-24

Susan K. Rathe

Tracy A. Marko

Elizabeth N. Edwards

Paige Hazelton Ridder

DOI：10.3390/genes16040369

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摘要

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利用CRISPR技术敲低或敲除基因是理解基因在疾病发生中特定作用的有力工具。然而，它可能引发许多未预期的转录组变化，这些变化无法通过靶位点DNA扩增和Sanger测序检测到。多种RNA测序技术可用于识别这些变化，并有效评估CRISPR敲除的全面影响，从而为筛选适合进一步实验的克隆提供方法。背景/目标：分析了4项CRISPR敲除实验的RNA-seq数据，并开发了用于确认CRISPR修饰成功及识别潜在问题的技术。方法：对RNA测序数据的广泛分析发现许多基于CRISPR的变化，这些变化在CRISPR靶位点周围DNA的PCR扩增中未被识别。这些变化包括染色体间融合事件、外显子跳读、染色体截断以及邻近基因的意外转录修饰和扩增。结论：通过评估4项CRISPR实验识别的意外修饰，突显了使用RNA-seq识别由CRISPR改变的细胞转录变化的价值，其中许多变化仅通过评估DNA无法识别。提出了利用RNA-seq数据设计和分析CRISPR实验的具体指南。

Keyword:

CRISPR

RNA sequencing

knockout experiments

期刊

Genes

IF:

2.8

论文数:

3.0K

被引数:

3.5W

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