Non-coding regulatory sequences play essential roles in adjusting gene output to cellular needs and are thus critical to animal development and health. Numerous such sequences have been identified in mammalian genomes ranging from transcription factors binding motifs to recognition sites for RNA-binding proteins and non-coding RNAs. The advent of CRISPR has raised the possibility of assigning functionality to individual endogenous regulatory sites by facilitating the generation of isogenic cell lines that differ by a defined set of genetic modifications. Here we investigate the usefulness of this approach to assign function to individual miRNA binding sites. We find that the process of generating isogenic pairs of mammalian cell lines with CRISPR-mediated mutations introduces extensive molecular and phenotypic variability between biological replicates confounding attempts at assigning function to the binding site. Our work highlights an important consideration when employing CRISPR editing to characterize non-coding regulatory sequences in cell lines and calls for the development and adoption of alternative strategies to address this question in the future.

中文翻译：

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使用同基因细胞系对 miRNA 结合位点进行基于 CRISPR 的解剖受到普遍噪声的阻碍


非编码调控序列在根据细胞需求调整基因输出方面起着至关重要的作用，因此对动物发育和健康至关重要。在哺乳动物基因组中已经鉴定出许多这样的序列，从转录因子结合基序到 RNA 结合蛋白和非编码 RNA 的识别位点。CRISPR 的出现通过促进产生因一组确定的基因修饰而不同的同基因细胞系，提高了将功能分配给单个内源性调节位点的可能性。在这里，我们研究了这种方法将功能分配给单个 miRNA 结合位点的有用性。我们发现，产生具有 CRISPR 介导突变的哺乳动物细胞系的同基因对的过程在生物重复之间引入了广泛的分子和表型变异性，混淆了将功能分配给结合位点的尝试。我们的工作突出了在使用 CRISPR 编辑来表征细胞系中的非编码调节序列时的一个重要考虑因素，并呼吁在未来开发和采用替代策略来解决这个问题。